Nucleic acids encoding human calcium channel and methods of use thereof

ABSTRACT

Isolated DNA encoding each of human calcium channel α 1 -, α 2 -, β- and γ-subunits, including subunits that arise as splice variants of primary transcripts, is provided. Cells and vectors containing the DNA and methods for identifying compounds that modulate the activity of human calcium channels are also provided.

This is a continuation of application Ser. No. 08/105,536, filed Aug.11, 1993, now abandoned which is a continuation-in-part of InternationalPCT application Serial No. PCT/US92/06903, filed Aug. 14, 1997, now U.S.patent application Ser. No. 08/193,078, filed Feb. 7, 1994, now U.S.Pat. No. 5,846,757, which is a continuation-in-part of U.S. applicationSer. No. 07/868,354, filed Apr. 10, 1992, now abandoned which is acontinuation-in-part of U.S. application Ser. No. 07/745,206, filed Aug.15, 1991, now U.S. Pat. No. 5,629,921 which is a continuation-in-part ofU.S. application Ser. No. 07/620,250, filed Nov. 30, 1990, nowabandoned, which is a continuation-in-part of U.S. application Ser. No.07/176,899, filed Apr. 4, 1988, now abandoned, and is also acontinuation-in-part of U.S. Ser. No. 07/482,384, filed Feb. 20, 1990,now U.S. Pat. No. 5,386,025. This application is also acontinuation-in-part of U.S. Ser. No. 07/914,231, filed Jul. 13, 1992now U.S. Pat. No. 5,607,820, which in turn is a continuation of U.S.Ser. No. 07/603,751, filed Nov. 8, 1990, now abandoned. This applicationis also a continuation-in-part of International Application Serial No.PCT/US89/01408, filed Apr. 4, 1988. U.S. application Ser. No. 07/603,751is International Application Serial No. PCT/89/01408.

The subject matter of each of International PCT Application Ser. No.PCT/92/06903, U.S. Ser. No. 07/914,231, U.S. application Ser. No.07/868,354, U.S. application Ser. No. 07/745,206 U.S. application Ser.No. 07/620,250, U.S. application Ser. No. 07/603,751, U.S. applicationSer. No. 07/482,384, U.S. application Ser. No. 07/176,899 andInternational Application Ser. No. PCT/89/01408 is incorporated hereinin its entirety.

TECHNICAL FIELD

The present invention relates to molecular biology and pharmacology.More particularly, the invention relates to calcium channel compositionsand methods of making and using the same.

BACKGROUND OF THE INVENTION

Calcium channels are membrane-spanning, multi-subunit proteins thatallow controlled entry of Ca²⁺ ions into cells from the extracellularfluid. Cells throughout the animal kingdom, and at least some bacterial,fungal and plant cells, possess one or more types of calcium channel.

The most common type of calcium channel is voltage dependent. “Opening”of a voltage-dependent channel to allow an influx of Ca²⁺ ions into thecells requires a depolarization to a certain level of the potentialdifference between the inside of the cell bearing the channel and theextracellular medium bathing the cell. The rate of influx of Ca²⁺ intothe cell depends on this potential difference. All “excitable” cells inanimals, such as neurons of the central nervous system (CNS), peripheralnerve cells and muscle cells, including those of skeletal muscles,cardiac muscles, and venous and arterial smooth muscles, havevoltage-dependent calcium channels.

Multiple types of calcium channels have been identified in mammaliancells from various tissues, including skeletal muscle, cardiac muscle,lung, smooth muscle and brain, [see, e.g., Bean, B. P. (1989) Ann. Rev.Physiol. 51:367-384 and Hess, P. (1990) Ann. Rev. Neurosci. 56:337]. Thedifferent types of calcium channels have been broadly categorized intofour classes, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists.

Calcium channels are multisubunit proteins. For example, rabbit skeletalmuscle calcium channel contains two large subunits, designated α₁ andα₂, which have molecular weights between about 130 and about 200kilodaltons (“kD”), and one to three different smaller subunits of lessthan about 60 kD in molecular weight. At least one of the largersubunits and possibly some of the smaller subunits are glycosylated.Some of the subunits are capable of being phosphorylated. The α₁ subunithas a molecular weight of about 150 to about 170 kD when analyzed bysodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)after isolation from mammalian muscle tissue and has specific bindingsites for various 1,4-dihydropyridines (DHPs) and phenylalkylamines.Under non-reducing conditions (in the presence of N-ethylmaleimide), theα₂ subunit migrates in SDS-PAGE as a band corresponding to a molecularweight of about 160-190 kD. Upon reduction, a large fragment and smallerfragments are released. The β subunit of the rabbit skeletal musclecalcium channel is a phosphorylated protein that has a molecular weightof 52-65 kD as determined by SDS-PAGE analysis. This subunit isinsensitive to reducing conditions. The γ subunit of the calciumchannel, which is not observed in all purified preparations, appears tobe a glycoprotein with an apparent molecular weight of 30-33 kD, asdetermined by SDS-PAGE analysis.

In order to study calcium channel structure and function, large amountsof pure channel protein are needed. Because of the complex nature ofthese multisubunit proteins, the varying concentrations of calciumchannels in tissue sources of the protein, the presence of mixedpopulations of calcium channels in tissues, difficulties in obtainingtissues of interest, and the modifications of the native protein thatcan occur during the isolation procedure, it is extremely difficult toobtain large amounts of highly purified, completely intact calciumchannel protein.

Characterization of a particular type of calcium channel by analysis ofwhole cells is severely restricted by the presence of mixed populationsof different types of calcium channels in the majority of cells.Single-channel recording methods that are used to examine individualcalcium channels do not reveal any information regarding the molecularstructure or biochemical composition of the channel. Furthermore, inperforming this type of analysis, the channel is isolated from othercellular constituents that might be important for natural functions andpharmacological interactions.

Characterization of the gene or genes encoding calcium channels providesanother means of characterization of different types of calciumchannels. The amino acid sequence determined from a complete nucleotidesequence of the coding region of a gene encoding a calcium channelprotein represents the primary structure of the protein. Furthermore,secondary structure of the calcium channel protein and the relationshipof the protein to the membrane may be predicted based on analysis of theprimary structure. For instance, hydropathy plots of the α₁ subunitprotein of the rabbit skeletal muscle calcium channel indicate that itcontains four internal repeats, each containing six putativetransmembrane regions [Tanabe, T. et al. (1987) Nature 328:313].

The cDNA and corresponding amino acid sequences of the α₁, α₂, β and γsubunits of the rabbit skeletal muscle calcium channel [see, Tanabe etal. (1987) Nature 328:313-318; International Application No. WO89/09834, which is U.S. application Ser. No. 07/603,751, which is acontinuation-in-part of U.S. application Ser. No. 07/176,899; Ruth etal. (1989) Science 245:1115-1118; and U.S. patent application Ser. No.482,384, filed Feb. 20, 1990] have been determined. The cDNA andcorresponding amino acid sequences of α₁ subunits of rabbit cardiacmuscle [Mikami, A. et al. (1989) Nature 340:230-233] and lung [Biel, M.(1990) FEBS Letters 269:409-412] calcium channels have been determined.

In addition, a cDNA clone encoding a rabbit brain calcium channel(designated the BI channel) has been isolated [Mori, Y. et al. (1991)Nature 350:398-402). Partial cDNA clones encoding portions of severaldifferent subtypes, referred to as rat brain class A, B, C and D, of thecalcium channel α₁ subunit have been isolated from rat brain cDNAlibraries (Snutch, T. et al. (1990) Proc. Natl. Acad. Sci. USA87:3391-3395]. More recently full-length rat brain class A [Starr, T. etal. (1991) Proc. Natl. Acad. Sci. USA 88:5621-5625] and class C [Snutch,T. et al. (1991) Neuron 7:45-57] cDNA clones have been isolated.Although the amino acid sequence encoded by the rat brain class C DNA isapproximately 95% identical to that encoded by the rabbit cardiac musclecalcium channel α₁ subunit-encoding DNA, the amino acid sequence encodedby the rat brain class A DNA shares only 33% sequence identity with theamino acid sequence encoded by the rabbit skeletal or cardiac muscle α₁subunit-encoding DNA. A cDNA clone encoding another rat brain calciumchannel α₁ subunit has also been obtained [Hui, A. et al. (1991) Neuron7:35-44]. The amino acid sequence encoded by this clone is ˜70%homologous to the proteins encoded by the rabbit skeletal and cardiacmuscle calcium channel DNA. A cDNA clone closely related to the ratbrain class C α₁ subunit-encoding cDNA and sequences of partial cDNAclones closely related to other partial cDNA clones encoding apparentlydifferent calcium channel α₁ subunits have also been isolated [seeSnutch, T. et al. (1991) Neuron 7:45-57; Perez-Reyes, E. et al. (1990)J. Biol. Chem. 265:20430; and Hui, A. et al. (1991) Neuron 7:35-44). DNAclones encoding other calcium channels have also been identified andisolated.

Expression of cDNA encoding calcium channel subunits has been achievedwith several of the different rabbit or rat α₁ subunit cDNA clonesdiscussed above. Voltage-dependent calcium currents have been detectedin murine L cells transfected with DNA encoding the rabbit skeletalmuscle calcium channel α₁ subunit [Perez-Reyes et al. (1989) Nature340:233-236 (1989)]. These currents were enhanced in the presence of thecalcium channel agonist Bay K 8644. Bay K 8644-sensitive Ba⁺ currentshave been detected in oöcytes injected with in vitro transcripts of therabbit cardiac muscle calcium channel α₁ subunit cDNA [Mikami, A. et al.(1989) Nature 340:230-233]. These currents were substantially reduced inthe presence of the calcium channel antagonist nifedipine. Bariumcurrents of an oöcyte co-injected with RNA encoding the rabbit cardiacmuscle calcium channel α₁ subunit and the RNA encoding the rabbitskeletal muscle calcium channel α₂ subunit were more than 2-fold largerthan those of oöcytes injected with transcripts of the rabbit cardiaccalcium channel α₁ subunit-encoding cDNA. Similar results were obtainedwhen oöcytes were co-injected with RNA encoding the rabbit lung calciumchannel α₁ subunit and the rabbit skeletal muscle calcium channel α₂subunit. The barium current was greater than that detected in oöcytesinjected only with RNA encoding the rabbit lung calcium channel α₁subunit [Biel, M. et al. (1990) FEBS Letters 269:409-412]. Inward bariumcurrents have been detected in oöcytes injected with in vitro RNAtranscripts encoding the rabbit brain BI channel [Mori et al. (1991)Nature 350:398-402]. These currents were increased by two orders ofmagnitude when in vitro transcripts of the rabbit skeletal musclecalcium channel α₂, β, or α₂, β and γ subunits were co-injected withtranscripts of the BI-encoding cDNA. Barium currents in oöcytesco-injected with transcripts encoding the BI channel and the rabbitskeletal muscle calcium channel α₂ and β were unaffected by the calciumchannel antagonists nifedipine or ω-CgTx and inhibited by Bay K 8644 andcrude venom from Agelenopsis aperta.

The results of studies of recombinant expression of rabbit calciumchannel α₁ subunit-encoding cDNA clones and transcripts of the cDNAclones indicate that the α₁ subunit forms the pore through which calciumenters cells. The relevance of the barium currents generated in theserecombinant cells to the actual current generated by calcium channelscontaining as one component the respective α₁ subunits in vivo isunclear. In order to completely and accurately characterize and evaluatedifferent calcium channel types, however, it is essential to examine thefunctional properties of recombinant channels containing all of thesubunits as found in vivo.

Although there has been limited success in expressing DNA encodingrabbit and rat calcium channel subunits, far less has been achieved withrespect to human calcium channels. Little is known about human calciumchannel structure and function and gene expression. An understanding ofthe structure and function of human calcium channels would permitidentification of substances that, in some manner, modulate the activityof calcium channels and that have potential for use in treating suchdisorders.

Because calcium channels are present in various tissues and have acentral role in regulating intracellular calcium ion concentrations,they are implicated in a number of vital processes in animals, includingneurotransmitter release, muscle contraction, pacemaker activity, andsecretion of hormones and other substances. These processes appear to beinvolved in numerous human disorders, such as CNS and cardiovasculardiseases. Calcium channels, thus, are also implicated in numerousdisorders. A number of compounds useful for treating variouscardiovascular diseases in animals, including humans, are thought toexert their beneficial effects by modulating functions ofvoltage-dependent calcium channels present in cardiac and/or vascularsmooth muscle. Many of these compounds bind to calcium channels andblock, or reduce the rate of, influx of Ca²⁺ into the cells in responseto depolarization of the cell membrane.

An understanding of the pharmacology of compounds that interact withcalcium channels in other organ systems, such as the CNS, may aid in therational design of compounds that specifically interact with subtypes ofhuman calcium channels to have desired therapeutic effects, such as inthe treatment of neurodegenerative and cardiovascular disorders. Suchunderstanding and the ability to rationally design therapeuticallyeffective compounds, however, have been hampered by an inability toindependently determine the types of human calcium channels and themolecular nature of individual subtypes, particularly in the CNS, and bythe unavailability of pure preparations of specific channel subtypes touse for evaluation of the specificity of calcium channel-effectingcompounds. Thus, identification of DNA encoding human calcium channelsubunits and the use of such DNA for expression of calcium channelsubunits and functional calcium channels would aid in screening anddesigning therapeutically effective compounds.

Therefore, it is an object herein, to provide DNA encoding specificcalcium channel subunits and to provide eukaryotic cells bearingrecombinant tissue-specific or subtype-specific calcium channels. It isalso an object to provide assays for identification of potentiallytherapeutic compounds that act as calcium channel antagonists andagonists.

SUMMARY OF THE INVENTION

Isolated and purified DNA fragments that encode human calcium channelsubunits are provided. DNA encoding α₁ subunits of a human calciumchannel, and RNA, encoding such subunits, made upon transcription ofsuch DNA are provided. In particular, DNA fragments encoding α₁ subunitsof voltage-dependent human calcium channels (VDCCs) type A, type B (alsoreferred to as VDCC IV), type C (also referred to as VDCC II) type D(also referred to as VDCC III) and type E are provided.

DNA encoding α_(1A), α_(1B), α_(1C), α_(1D) and α_(1E) subunits isprovided. DNA encoding an α_(1D) subunit that includes the amino acidssubstantially as set forth as residues 10-2161 of SEQ ID No. 1 isprovided. DNA encoding an α_(1D) subunit that includes substantially theamino acids set forth as amino acids 1-34 in SEQ ID No. 2 in place ofamino acids 373-406 of SEQ ID No. 1 is also provided. DNA encoding anα_(1C) subunit that includes the amino acids substantially as set forthin SEQ ID No. 3 or SEQ ID No. 6 and DNA encoding an α_(1B) subunit thatincludes an amino acid sequence substantially as set forth in SEQ ID No.7 or in SEQ ID No. 8 is also provided.

DNA encoding α_(1A) subunits is also provided. Such DNA includes DNAencoding an α_(1A) subunit that has substantially the same sequence ofamino acids as that set forth in SEQ ID No. 22 or No. 23 or other splicevariants of α_(1A) that include all or part of the sequence set forth inSEQ ID No. 22 or 23. The sequence set forth in SEQ ID NO. 22 is a splicevariant designated α_(1A-1); and the sequence set forth in SEQ ID NO. 23is a splice variant designated α_(1A-2). α_(1A) subunits also includesubunits that can be isolated using all or a portion of the DNA havingSEQ ID NO. 21, 22 or 23 or DNA obtained from the phage lysate of an E.coli host containing DNA encoding an α_(1A) subunit that has beendeposited in the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852 U.S.A. under Accession No. 75293 in accord with theBudapest Treaty. The DNA in such phage includes a DNA fragment havingthe sequence set forth in SEQ ID No. 21. This fragment selectivelyhybridizes under conditions of high stringency to DNA encoding α_(1A)but not to DNA encoding α_(1B) and, thus, can be used to isolate DNAthat encodes α_(1A) subunits.

DNA encoding α_(1E) subunits of a human calcium channel is alsoprovided. This DNA includes DNA that encodes an α_(1E) splice variantdesignated α_(1E-1) encoded by the DNA set forth in SEQ ID No. 24, and avariant designated α_(1E-3) encoded by SEQ. ID No. 24 with the fragmentset forth in SEQ ID No. 25 inserted between nucleotides 2405 and 2406.The resulting sequence of α_(1E-3) is set forth in SEQ ID No. 27. ThisDNA also includes other splice variants thereof that include sequencesof amino acids encoded by all or a portion of the sequences ofnucleotides set forth in SEQ ID Nos. 24 and 25 and DNA that hybridizesunder conditions of high stringency to the DNA of SEQ ID. No. 24 or 25and that encodes an α_(1E) splice variant.

DNA encoding α₂ subunits of a human calcium channel, and RNA encodingsuch subunits, made upon transcription of such a DNA are provided. DNAencoding splice variants of the α₂ subunit, including tissue specificsplice variants, are also provided. In particular, DNA encoding theα_(2a)-α_(2c) subunit subtypes is provided. In particularly preferredembodiments, the DNA encoding the α₂ subunit is produced by alternativeprocessing of a primary transcript that includes DNA encoding the aminoacids set forth in SEQ ID 11 and the DNA of SEQ ID No. 13 insertedbetween nucleotides 1624 and 1625 of SEQ ID No. 11.

The resulting sequences of each of the α₂-subunits are set forth inSequence ID NO. 11 (α_(2b)), SEQ ID NO. 28 (α_(2a)), SEQ ID NO. 29(α_(2c)), SEQ ID NO. 30 (α_(2d)), an d SEQ ID NO. 31 (α_(2e)).

Isolated and purified DNA fragments encoding human calcium channel βsubunits, including DNA encoding β₁ and β₂ subunit splice variants andthe β₃ subunit is provided. RNA encoding β subunits, made upontranscription of the DNA is also provided. In particular, DNA encodingthe β₁, β₂ and β₃ subunits, including the β₁ subunit splice variantsβ₁₋₁-β₁₋₅, described below, the β₂ subunit splice variantsβ_(2A)-β_(2E), that include all or a portion of SEQ ID No. 26, and theβ₃ subunit, that includes sequence set forth in SEQ ID Nos 19 and 20, isprovided. Escherichia coli (E. coli) host cells harboring plasmidscontaining DNA encoding β₃ have been deposited in accord with theBudapest Treaty under Accession No. 69048 at the American Type CultureCollection. A partial sequence of the deposited clone is set forth inSEQ ID No. 19 (sequence from the 5′ end) and SEQ ID No. 20 (sequencefrom the 3′ end).

DNA encoding β₁ subunits that are produced by alternative processing ofa primary transcript encoding a β subunit, including a transcript thatincludes DNA encoding the amino acids set forth in SEQ ID No. 9 orincluding a primary transcript that encodes β₃ as deposited under ATCCAccession No. 69048, but lacking and including alternative exons areprovided or may be constructed from the DNA provided herein. Forexample, DNA encoding a β₁ subunit that is produced by alternativeprocessing of a primary transcript that includes DNA encoding the aminoacids set forth in SEQ ID No. 9, but including the DNA set forth in SEQID No. 12 inserted in place of nucleotides 615-781 of SEQ ID No. 9 isalso provided. DNA encoding β₁ subunits that are encoded by transcriptsthat have the sequence set forth in SEQ ID No. 9 including the DNA setforth in SEQ ID No. 12 inserted in place of nucleotides 615-781 of SEQID No. 9, but that lack one or more of the following sequences ofnucleotides: nucleotides 14-34 of SEQ ID No. 12, nucleotides 13-34 ofSEQ ID No. 12, nucleotides 35-55 of SEQ ID No 12, nucleotides 56-190 ofSEQ ID No. 12 and nucleotides 191-271 of SEQ ID No. 12 are alsoprovided.

DNA encoding γ subunits of human calcium channels is also provided. RNA,encoding γ subunits, made upon transcription of the DNA are alsoprovided. In particular, DNA containing the sequence of nucleotides setforth in SEQ ID No. 14 is provided.

Full-length DNA clones and corresponding RNA transcripts, encoding theα₁, including splice variants of α_(1D), α_(1B), and α_(1E), α₂ and βsubunits, including β₁₋₁-β₁₋₅ and β_(2D) of human calcium channels areprovided. Also provided are DNA clones encoding substantial portions ofthe α_(1A), α_(1C), β₃ and γ subunits of voltage-dependent human calciumchannels for the preparation of full-length DNA clones encoding thefull-length α_(1A), α_(1c), β₃ and γ subunits.

Eukaryotic cells containing heterologous DNA encoding one or morecalcium channel subunits, particularly human calcium channel subunits,or containing RNA transcripts of DNA clones encoding one or more of thesubunits are provided. In preferred embodiments, the cells contain DNAor RNA encoding a human α₁ subunit, preferably at least an α_(1D),α_(1B) or α_(1E) subunit. In more preferred embodiments, the cellscontain DNA or RNA encoding additional heterologous subunits, includingat least one β, α₂ or γ subunits. In such embodiments, eukaryotic cellsstably or transiently transfected with any combination of one, two,three or four of the subunit-encoding DNA clones, such as DNA encodingany of α₁, α₁+β, α₁+β+α₂, are provided.

In preferred embodiments, the cells express such heterologous calciumchannel subunits and include one or more of the subunits in membranespanning heterologous calcium channels. In more preferred embodiments,the eukaryotic cells express functional, heterologous calcium channelsthat are capable of gating the passage of calcium channel selective ionsand/or binding compounds that, at physiological concentrations, modulatethe activity of the heterologous calcium channel. In certainembodiments, the heterologous calcium channels include at least oneheterologous calcium channel subunit. In most preferred embodiments, thecalcium channels that are expressed on the surface of the eukaryoticcells are composed substantially or entirely of subunits encoded by theheterologous DNA or RNA. In preferred embodiments, the heterologouscalcium channels of such cells are distinguishable from any endogenouscalcium channels of the host cell. Such cells provide a means to obtainhomogeneous populations of calcium channels.

In certain embodiments the recombinant eukaryotic cells that contain theheterologous DNA encoding the calcium channel subunits are produced bytransfection with DNA encoding one or more of the subunits or areinjected with RNA transcripts of DNA encoding one or more of the calciumchannel subunits. The DNA may be introduced as a linear DNA fragment ormay be included in an expression vector for stable or transientexpression of the subunit-encoding DNA. Vectors containing DNA encodinghuman calcium channel subunits are also provided.

The eukaryotic cells that express heterologous calcium channels may beused in assays for calcium channel function or, in the case of cellstransformed with fewer subunit-encoding nucleic acids than necessary toconstitute a functional recombinant human calcium channel, such cellsmay be used to assess the effects of additional subunits on calciumchannel activity. The additional subunits can be provided bysubsequently transfecting such a cell with one or more DNA clones or RNAtranscripts encoding human calcium channel subunits.

The recombinant eukaryotic cells that express membrane spanningheterologous calcium channels may be used in methods for identifyingcompounds that modulate calcium channel activity. In particular, thecells are used in assays that identify agonists and antagonists ofcalcium channel activity in humans and/or assessing the contribution ofthe various calcium channel subunits to the transport and regulation oftransport of calcium ions. Because the cells constitute homogeneouspopulations of calcium channels, they provide a means to identifyagonists or antagonists of calcium channel activity that are specificfor each such population.

The assays that use the eukaryotic cells for identifying compounds thatmodulate calcium channel activity are also provided. In practicing theseassays the eukaryotic cell that expresses a heterologous calciumchannel, containing at least on subunit encoded by the DNA providedherein, is in a solution containing a test compound and a calciumchannel selective ion, the cell membrane is depolarized, and currentflowing into said cell is detected. The current that is detected isdifferent from that produced by depolarizing the same or a substantiallyidentical cell in the presence of the same calcium channel selective ionbut in the absence of said compound. In preferred embodiments, prior tothe depolarization step, the cell is maintained at a holding potentialwhich substantially inactivates calcium channels which are endogenous tosaid cell. Also in preferred embodiments, the cells are mammalian cells,most preferably HEK cells, or amphibian oöcytes.

Nucleic acid probes containing at least about 14 contiguous nucleotidesof α_(1D), α_(1C), α_(1B), α_(1A) and α_(1E), α₂, β, including β₁ and β₂splice variants and β₃, and γ subunit-encoding DNA are provided. Methodsusing the probes for the isolation and cloning of calcium channelsubunit-encoding DNA, including splice variants within tissues andinter-tissue variants are also provided.

Purified human calcium channel subunits and purified human calciumchannels are provided. The subunits and channels can be isolated from aeukaryotic cell transfected with DNA that encodes the subunit.

In another embodiment, immunoglobulins or antibodies obtained from theserum of an animal immunized with a substantially pure preparation of ahuman calcium channel, human calcium channel subunit orepitope-containing fragment of a human calcium subunit are provided.Monoclonal antibodies produced using a human calcium channel, humancalcium channel subunit or epitope-containing fragment thereof as animmunogen are also provided. E. coli fusion proteins including afragment of a human calcium channel subunit may also be used asimmunogen. Such fusion proteins may contain a bacterial protein orportion thereof, such as the E. coli TrpE protein, fused to a calciumchannel subunit peptide. The immunoglobulins that are produced using thecalcium channel subunits or purified calcium channels as immunogenshave, among other properties, the ability to specifically andpreferentially bind to and/or cause the immunoprecipitation of a humancalcium channel or a subunit thereof which may be present in abiological sample or a solution derived from such a biological sample.Such antibodies may also be used to selectively isolate cells thatexpress calcium channels that contain the subunit for which theantibodies are specific.

A diagnostic method for determining the presence of Lambert EatonSyndrome (LES) in a human based on immunological reactivity of LESimmunoglobulin G (IgG) with a human calcium channel subunit or aeukaryotic cell which expresses a recombinant human calcium channel or asubunit thereof is also provided. In particular, an immunoassay methodfor diagnosing Lambert-Eaton Syndrome in a person by combining serum oran IgG fraction from the person (test serum) with calcium channelproteins, including the α and 62 subunits, and ascertaining whetherantibodies in the test serum react with one or more of the subunits, ora recombinant cell which expresses one or more of the subunits to agreater extent than antibodies in control serum, obtained from a personor group of persons known to be free of the Syndrome, is provided. Anyimmunoassay procedure known in the art for detecting antibodies againsta given antigen in serum can be employed in the method.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference herein.

Reference to each of the calcium channel subunits includes the subunitsthat are specifically disclosed herein and human calcium channelsubunits encoded by DNA that can be isolated by using the DNA disclosedas probes and screening an appropriate human cDNA or genomic libraryunder at least low stringency. Such DNA also includes DNA that encodesproteins that have about 40% homology to any of the subunits proteinsdescribed herein or DNA that hybridizes under conditions of at least lowstringency to the DNA provided herein and the protein encoded by suchDNA exhibits additional identifying characteristics, such as function ormolecular weight.

It is understood that subunits that are encoded by transcripts thatrepresent splice variants of the disclosed subunits or other suchsubunits may exhibit less than 40% overall homology to any singlesubunit, but will include regions of such homology to one or more suchsubunits. It is also understood that 40% homology refers to proteinsthat share approximately 40% of their amino acids in common or thatshare somewhat less, but include conservative amino acid substitutions,whereby the activity of the protein is not substantially altered.

As used herein, the α₁ subunits types, encoded by different genes, aredesignated as type α_(1A), α_(1B), α_(1C), α_(1D) and α_(1E). Thesetypes may also be also referred to as VDCC IV for α_(1B), VDCC II forα_(1C) and VDCC III for α_(1D). Subunit subtypes, which are splicevariants, are referred to, for example as α_(1B-1), α_(1B-2), α_(1C-1)etc.

Thus, as used herein, DNA encoding the α₁ subunit refers to DNA thathybridizes to the DNA provided herein under conditions of at least lowstringency or encodes a subunit that has roughly about 40% homology toprotein encoded by DNA disclosed herein that encodes an α₁ subunit of ahuman calcium. An α₁ subunit may be identified by its ability to form acalcium channel. Typically, α₁ subunits have molecular weights greaterthan at least about 120 kD. The activity of a calcium channel may beassessed in vitro by methods known to those of skill in the art,including the electrophysiological and other methods described herein.Typically, α₁ subunits include regions to which one or more modulatorsof calcium channel activity, such as a 1,4 DHP or ω-CgTX, interactdirectly or indirectly. Types of α₁ subunits may be distinguished by anymethod known to those of skill in the art, including on the basis ofbinding specificity. For example, it has been found herein that α_(1B)subunits participate in the formation channels that have previously beenreferred to as N-type channels, α_(1D) subunits participate in theformation of channels that had previously been referred to as L-typechannels, and α_(1A) subunits appear to participate in the formation ofchannels that exhibit characteristics typical of channels that hadpreviously been designated P-type channels. Thus, for example, theactivity of channels that contain the α_(1B) subunit are insensitive to1,4 DHPs; whereas the activity of channels that contain the α_(1D)subunit are modulated or altered by a 1,4 DHP. It is presentlypreferable to refer to calcium channels based on pharmacologicalcharacteristics and current kinetics and to avoid historicaldesignations. Types and subtypes of α₁ subunits may be characterized onthe basis of the effects of such modulators on the subunit or a channelcontaining the subunit as well as differences in currents and currentkinetics produced by calcium channels containing the subunit.

As used herein, an α₂ subunit is encoded by DNA that hybridizes to theDNA provided herein under conditions of low stringency or encodes aprotein that has about 40% homology with that disclosed herein. Such DNAencodes a protein that typically has a molecular weight greater thanabout 120 kD, but does not form a calcium channel in the absence of anα₁ subunit, and may alter the activity of a calcium channel thatcontains an α₁ subunit. Subtypes of the α₂ subunit that arise as splicevariants are designated by lower case letter, such as α_(2a), . . .α_(2e). In addition, the α₂ subunit and the large fragment producedunder reducing conditions appear to be glycosylated with at leastN-linked sugars and do not specifically bind to the 1,4-DHPs andphenylalkylamines that specifically bind to the α₁ subunit. The smallerfragment, the C-terminal fragment, is referred to as the δ subunit andincludes amino acids from about 946 (SEQ ID No. 11) through about theC-terminus. This fragment may dissociate from the remaining portion ofα₂ when the α₂ subunit is exposed to reducing conditions.

As used herein, a β subunit is encoded by DNA that hybridizes to the DNAprovided herein under conditions of low stringency or encodes a proteinthat has about 40% homology with that disclosed herein and is a proteinthat typically has a molecular weight lower than the a subunits and onthe order of about 50-80 kD, does not form a detectable calcium channelin the absence of an α₁ subunit, but may alter the activity of a calciumchannel that contains an α₁ subunit or that contains an α₁ and α₂subunit.

Types of the β subunit that are encoded by different genes aredesignated with subscripts, such as β₁, β₂ and β₃. Subtypes of βsubunits that arise as splice variants of a particular type aredesignated with a numerical subscript referring to the subtype and tothe variant. Such subtypes include, but are not limited to the β₁ splicevariants, including β₁₋₁-β₁₋₅ and β₂ variants, including β_(2A)-β_(2E).

As used herein, a γ subunit is a subunit encoded by DNA disclosed hereinas encoding the γ subunit and may be isolated and identified using theDNA disclosed herein as a probe by hybridization or other such methodknown to those of skill in the art, whereby full-length clones encodinga γ subunit may be isolated or constructed. A γ subunit will be encodedby DNA that hybridizes to the DNA provided herein under conditions oflow stringency or exhibits sufficient sequence homology to encode aprotein that has about 40% homology with the γ subunit described herein.

Thus, one of skill in the art, in light of the disclosure herein, canidentify DNA encoding α₁, α₂, β, δ and calcium channel subunits,including types encoded by different genes and subtypes that representsplice variants. For example, DNA probes based on the DNA disclosedherein may be used to screen an appropriate library, including a genomicor cDNA library, and obtain DNA in one or more clones that includes anopen reading fragment that encodes an entire protein. Subsequent toscreening an appropriate library with the DNA disclosed herein, theisolated DNA can be examined for the presence of an open reading framefrom which the sequence of the encoded protein may be deduced.Determination of the molecular weight and comparison with the sequencesherein should reveal the identity of the subunit as an α₁, α₂ etc.subunit. Functional assays may, if necessary, be used to determinewhether the subunit is an α₁, α₂ subunit or β subunit.

For example, DNA encoding an α_(1A) subunit may be isolated by screeningan appropriate library with DNA, encoding all or a portion of the humanα_(1A) subunit. Such DNA includes the DNA in the phage deposited underATCC Accession No. 75293 that encodes an α₁ subunit. DNA encoding anα_(1A) subunit may obtained from an appropriate library by screeningwith an oligonucleotide having all or a portion of the sequence setforth in SEQ ID No, 21, 22 and/or 23 or with the DNA in the depositedphage. Alternatively, such DNA may have a sequence that encodes anα_(1A) subunit that is encoded by SEQ ID NO. 22 or 23.

Similarly, DNA encoding β₃ may be isolated by screening a human cDNAlibrary with DNA probes prepared from the plasmid β1.42 deposited underATCC Accession No. 69048 or obtained from an appropriate library usingprobes having sequences prepared according to the sequences set forth inSEQ ID Nos. 19, 20 and/or 30. Any method known to those of skill in theart for isolation and identification of DNA and preparation offull-length genomic or cDNA clones, including methods exemplifiedherein, may be used.

The subunit encoded by isolated DNA may be identified by comparison withthe DNA and amino acid sequences of the subunits provided herein. Splicevariants share extensive regions of homology, but include non-homologousregions, subunits encoded by different genes share a uniformdistribution of non-homologous sequences.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA. Splice variants may occur withina single tissue type or among tissues (tissue-specific variants). Thus,cDNA clones that encode calcium channel subunit subtypes that haveregions of identical amino acids and regions of different amino acidsequences are referred to herein as “splice variants”.

As used herein, a “calcium channel selective ion” is an ion that iscapable of flowing through, or being blocked from flowing through, acalcium channel which spans a cellular membrane under conditions whichwould substantially similarly permit or block the flow of Ca²⁺. Ba⁺ isan example of an ion which is a calcium channel selective ion.

As used herein, a compound that modulates calcium channel activity isone that affects the ability of the calcium channel to pass calciumchannel selective ions or affects other detectable calcium channelfeatures, such as current kinetics. Such compounds include calciumchannel antagonists and agonists and compounds that exert their effecton the activity of the calcium channel directly or indirectly.

As used herein, a “substantially pure” subunit or protein is a subunitor protein that is sufficiently free of other polypeptide contaminantsto appear homogeneous by SDS-PAGE or to be unambiguously sequenced.

As used herein, selectively hybridize means that a DNA fragmenthybridizes to second fragment with sufficient specificity to permit thesecond fragment to be identified or isolated from among a plurality offragments. In general, selective hybridization occurs at conditions ofhigh stringency.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome in which it is present or which is found in alocation or locations in the genome that differ from that in which itoccurs in nature. It is DNA or RNA that is not endogenous to the celland has been artificially introduced into the cell. Examples ofheterologous DNA include, but are not limited to, DNA that encodes acalcium channel subunit and DNA that encodes RNA or proteins thatmediate or alter expression of endogenous DNA by affectingtranscription, translation, or other regulatable biochemical processes.The cell that expresses the heterologous DNA, such as DNA encoding thecalcium channel subunit, may contain DNA encoding the same or differentcalcium channel subunits. The heterologous DNA need not be expressed andmay be introduced in a manner such that it is integrated into the hostcell genome or is maintained episomally.

As used herein, operative linkage of heterologous DNA to regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences, refers to the functional relationship between such DNA andsuch sequences of nucleotides. For example, operative linkage ofheterologous DNA to a promoter refers to the physical and functionalrelationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA in reading frame.

As used herein, isolated, substantially pure DNA refers to DNA fragmentspurified according to standard techniques employed by those skilled inthe art (see, e.g., Maniatis et al. (1982) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.].

As used herein, expression refers to the process by which nucleic acidis transcribed into mRNA and translated into peptides, polypeptides, orproteins. If the nucleic acid is derived from genomic DNA, expressionmay, if an appropriate eukaryotic host cell or organism is selected,include splicing of the mRNA.

As used herein, vector or plasmid refers to discrete elements that areused to introduce heterologous DNA into cells for either expression ofthe heterologous DNA or for replication of the cloned heterologous DNA.Selection and use of such vectors and plasmids are well within the levelof skill of the art.

As used herein, expression vector includes vectors capable of expressingDNA fragments that are in operative linkage with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Thus, an expression vector refers to a recombinantDNA or RNA construct, such as a plasmid, a phage, recombinant virus orother vector that, upon introduction into an appropriate host cell,results in expression of the cloned DNA. Appropriate expression vectorsare well known to those of skill in the art and include those that arereplicable in eukaryotic cells and/or prokaryotic cells and those thatremain episomal or may integrate into the host cell genome.

As used herein, a promoter region refers to the portion of DNA of a genethat controls transcription of DNA to which it is operatively linked.The promoter region includes specific sequences of DNA that aresufficient for RNA polymerase recognition, binding and transcriptioninitiation. This portion of the promoter region is referred to as thepromoter. In addition, the promoter region includes sequences thatmodulate this recognition, binding and transcription initiation activityof the RNA polymerase. These sequences may be cis acting or may beresponsive to trans acting factors. Promoters, depending upon the natureof the regulation, may be constitutive or regulated.

As used herein, a recombinant eukaryotic cell is a eukaryotic cell thatcontains heterologous DNA or RNA.

As used herein, a recombinant or heterologous calcium channel refers toa calcium channel that contains one or more subunits that are encoded byheterologous DNA that has been introduced into and expressed in aeukaryotic cells that expresses the recombinant calcium channel. Arecombinant calcium channel may also include subunits that are producedby DNA endogenous to the cell. In certain embodiments, the recombinantor heterologous calcium channel may contain only subunits that areencoded by heterologous DNA.

As used herein, “functional” with respect to a recombinant orheterologous calcium channel means that the channel is able to providefor and regulate entry of calcium channel selective ions, including, butnot limited to, Ca²⁺ or Ba⁺, in response to a stimulus and/or bindligands with affinity for the channel. Preferably such calcium channelactivity is distinguishable, such as electrophysiological,pharmacological and other means known to those of skill in the art, fromany endogenous calcium channel activity that in the host cell.

As used herein, a peptide having an amino acid sequence substantially asset forth in a particular SEQ ID No. includes peptides that have thesame function but may include minor variations in sequence, such asconservative amino acid changes or minor deletions or insertions that donot alter the activity of the peptide. The activity of a calcium channelreceptor subunit peptide refers to its ability to form functionalcalcium channels with other such subunits.

As used herein, a physiological concentration of a compound is thatwhich is necessary and sufficient for a biological process to occur. Forexample, a physiological concentration of a calcium channel selectiveion is a concentration of the calcium channel selective ion necessaryand sufficient to provide an inward current when the channels open.

As used herein, activity of a calcium channel refers to the movement ofa calcium selective ion through a calcium channel. Such activity may bemeasured by any method known to those of skill in the art, including,but not limited to, measurement of the amount of current which flowsthrough the recombinant channel in response to a stimulus.

As used herein, a “functional assay” refers to an assay that identifiesfunctional calcium channels. A functional assay, thus, is an assay toassess function.

As understood by those skilled in the art, assay methods for identifyingcompounds, such as antagonists and agonists, that modulate calciumchannel activity, generally requires comparison to a control. One typeof a “control” cell or “control” culture is a cell or culture that istreated substantially the same as the cell or culture exposed to thetest compound except that the control culture is not exposed to the testcompound. Another type of a “control” cell or “control” culture may be acell or a culture of cells which are identical to the transfected cellsexcept the cells employed for the control culture do not expressfunctional calcium channels. In this situation, the response of testcell to the test compound compared to the response (or lack of response)of the receptor-negative cell to the test compound, when cells orcultures of each type of cell are exposed to substantially the samereaction conditions in the presence of the compound being assayed. Forexample, in methods that use patch clamp electrophysiologicalprocedures, the same cell can be tested in the presence and absence ofthe test compound, by changing the external solution bathing the cell asknown in the art.

Identification and Isolation of DNA Encoding Human Calcium ChannelSubunits

Methods for identifying and isolating DNA encoding α₁, α₂, β and γsubunits of human calcium channels are provided.

Identification and isolation of such DNA may be accomplished byhybridizing, under appropriate conditions, at least low stringencywhereby DNA that encodes the desired subunit is isolated, restrictionenzyme-digested human DNA with a labeled probe having at least 14nucleotides and derived from any contiguous portion of DNA having asequence of nucleotides set forth herein by sequence identificationnumber. Once a hybridizing fragment is identified in the hybridizationreaction, it can be cloned employing standard cloning techniques knownto those of skill in the art. Full-length clones may be identified bythe presence of a complete open reading frame and the identity of theencoded protein verified by sequence comparison with the subunitsprovided herein and by functional assays to assess calcium channelforming ability or other function. This method can be used to identifygenomic DNA encoding the subunit or cDNA encoding splice variants ofhuman calcium channel subunits generated by alternative splicing of theprimary transcript of genomic subunit DNA. For instance, DNA, cDNA orgenomic DNA, encoding a calcium channel subunit may be identified byhybridization to a DNA probe and characterized by methods known to thoseof skill in the art, such as restriction mapping and DNA sequencing, andcompared to the DNA provided herein in order to identify heterogeneityor divergence in the sequences the DNA. Such sequence differences mayindicate that the transcripts from which the cDNA was produced resultfrom alternative splicing of a primary transcript, if the non-homologousand homologous regions are clustered, or from a different gene if thenon-homologous regions are distributed throughout the cloned DNA.

Any suitable method for isolating genes using the DNA provided hereinmay be used. For example, oligonucleotides corresponding to regions ofsequence differences have been used to isolate, by hybridization, DNAencoding the full-length splice variant and can be used to isolategenomic clones. A probe, based on a nucleotide sequence disclosedherein, which encodes at least a portion of a subunit of a human calciumchannel, such as a tissue-specific exon, may be used as a probe to clonerelated DNA, to clone a full-length cDNA clone or genomic clone encodingthe human calcium channel subunit.

Labeled, including, but not limited to, radioactively or enzymaticallylabeled, RNA or single-stranded DNA of at least 14 substantiallycontiguous bases, preferably at least 30 contiguous bases of a nucleicacid which encodes at least a portion of a human calcium channelsubunit, the sequence of which nucleic acid corresponds to a segment ofa nucleic acid sequence disclosed herein by reference to a SEQ ID No.are provided. Such nucleic acid segments may be used as probes in themethods provided herein for cloning DNA encoding calcium channelsubunits. See, generally, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press.

In addition, nucleic acid amplification techniques, which are well knownin the art, can be used to locate splice variants of calcium channelsubunits by employing oligonucleotides based on DNA sequencessurrounding the divergent sequence primers for amplifying human RNA orgenomic DNA. Size and sequence determinations of the amplificationproducts can reveal splice variants. Furthermore, isolation of humangenomic DNA sequences by hybridization can yield DNA containing multipleexons, separated by introns, that correspond to different splicevariants of transcripts encoding human calcium channel subunits.

DNA encoding types and subtypes of each of the α₁, α₂, β and γ subunitof voltage-dependent human calcium channels has been cloned herein byscreening human cDNA libraries prepared from isolated poly A+ mRNA fromcell lines or tissue of human origin having such calcium channels. Amongthe sources of such cells or tissue for obtaining mRNA are human braintissue or a human cell line of neural origin, such as a neuroblastomacell line, human skeletal muscle or smooth muscle cells, and the like.Methods of preparing cDNA libraries are well known in the art [seegenerally Ausubel et al. (1987) Current Protocols in Molecular Biology,Wiley-Interscience, New York; and Davis et al. (1986) Basic Methods inMolecular Biology, Elsevier Science Publishing Co., New York].

With respect to each of the respective subunits of a human calciumchannel (α₁, α₂, β or γ), once the DNA encoding the channel subunit wasidentified by a nucleic acid screening method, the isolated clone wasused for further screening to identify overlapping clones. Some of thecloned DNA fragments can and have been subcloned into an appropriatevector such as pIBI24/25 (IBI, New Haven, Conn.), M13mp18/19, pGEM4,pGEM3, pGEM7Z, pSP72 and other such vectors known to those of skill inthis art, and characterized by DNA sequencing and restriction enzymemapping. A sequential series of overlapping clones may thus be generatedfor each of the subunits until a full-length clone can be prepared bymethods, known to those of skill in the art, that include identificationof translation initiation (start) and translation termination (stop)codons. For expression of the cloned DNA, the 5′ noncoding region andother transcriptional and translational control regions of such a clonemay be replaced with an efficient ribosome binding site and otherregulatory regions as known in the art. Other modifications of the 5′end, known to those of skill in the art, that may be required tooptimize translation and/or transcription efficiency may also beeffected, if deemed necessary.

Examples II-VI, below, describe in detail the cloning of each of thevarious subunits of a human calcium channel as well as subtypes andsplice variants, including tissue-specific variants thereof. In theinstances in which partial sequences of a subunit are disclosed, it iswell within the skill of the art, in view of the teaching herein, toobtain the corresponding full-length nucleotide sequence encoding thesubunit, subtype or splice variant thereof.

Identification and Isolation of DNA Encoding α₁ Subunits

A number of voltage-dependent calcium channel α₁ subunit genes, whichare expressed in the human CNS and in other tissues, have beenidentified and have been designated as α_(1A), α_(1B) (or VDCC IV),α_(1C) (or VDCC II), α_(1D) (or VDCC III) and α_(1E). DNA, isolated froma human neuronal cDNA library, that encodes each of the subunit typeshas been isolated. DNA encoding subtypes of each of the types, whicharise as splice variants are also provided. Subtypes are hereindesignated, for example, as α_(1B-1), α_(1B-2).

The α₁ subunits types A B, C, D and E of voltage-dependent calciumchannels, and subtypes thereof, differ with respect to sensitivity toknown classes of calcium channel agonists and antagonists, such as DHPs,phenylalkylamines, omega conotoxin (ω-CgTx), the funnel web spider toxinω-Aga-IV, and pyrazonoylguanidines. They also appear to differ in theholding potential and ion the kinetics of currents produced upondepolarization of cell membranes containing calcium channels thatinclude different types of α₁ subunits.

DNA that encodes an α₁-subunit that binds to at least one compoundselected from among dihydropyridines, phenylalkylamines, ω-CgTx,components of funnel web spider toxin, and pyrazonoylguanidines isprovided. For example, the α_(1B) subunit provided herein appears tospecifically interact with ω-CgTx in N-type channels, and the α_(1D)subunit provided herein specifically interacts with DHPs in L-typechannels.

Identification and Isolation of DNA Encoding the α_(1D) Human CalciumChannel Subunit

The α_(1D) subunit cDNA has been isolated using fragments of the rabbitskeletal muscle calcium channel α₁ subunit cDNA as a probe to screen acDNA library of a human neuroblastoma cell line, IMR32, to obtain cloneα1.36. This clone was used as a probe to screen additional IMR32 cellcDNA libraries to obtain overlapping clones, which were then employedfor screening until a sufficient series of clones to span the length ofthe nucleotide sequence encoding the human α_(1D) subunit were obtained.Full-length clones encoding α_(1D) were constructed by ligating portionsof partial α_(1D) clones as described in Example II. SEQ ID No. 1 showsthe 7,635 nucleotide sequence of the cDNA encoding the α_(1D) subunit.There is a 6,483 nucleotide sequence reading frame which encodes asequence of 2,161 amino acids (as set forth in SEQ ID No. 1).

SEQ ID No. 2 provides the sequence of an alternative exon encoding theIS6 transmembrane domain [see Tanabe, T., et al. (1987) Nature328:313-318 for a description of transmembrane domain terminology] ofthe α_(1D) subunit.

SEQ ID No. 1 also shows the 2,161 amino acid sequence deduced from thehuman neuronal calcium channel α_(1D) subunit DNA. Based on the aminoacid sequence, the α_(1D) protein has a calculated Mr of 245,163. Theα_(1D) subunit of the calcium channel contains four putative internalrepeated sequence regions. Four internally repeated regions represent 24putative transmembrane segments, and the amino- and carboxyl-terminiextend intracellularly.

The α_(1D) subunit has been shown to mediate DHP-sensitive,high-voltage-activated, long-lasting calcium channel activity. Thiscalcium channel activity was detected when oöcytes were co-injected withRNA transcripts encoding an α_(1D) and β₁ or α_(1D), α₂ and β₁ subunits.This activity was distinguished from Ba²⁺ currents detected when oöcyteswere injected with RNA transcripts encoding the β₁±α₂ subunits. Thesecurrents pharmacologically and biophysically resembled Ca²⁺ currentsreported for uninjected oöcytes.

Identification and Isolation DNA Encoding the α_(1A) Human CalciumChannel Subunit

Biological material containing DNA encoding the α_(1A) subunit had beendeposited in the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852 U.S.A. under the terms of the Budapest Treaty onthe International Recognition of Deposits of Microorganisms for Purposesof Patent Procedure and the Regulations promulgated under this Treaty.Samples of the deposited material are and will be available toindustrial property offices and other persons legally entitled toreceive them under the terms of said Treaty and Regulations andotherwise in compliance with the patent laws and regulations of theUnited States of America and all other nations or internationalorganizations in which this application, or an application claimingpriority of this application, is filed or in which any patent granted onany such application is granted.

An α_(1A) subunit is encoded by an approximately 3 kb insert in λgt10phage designated α1.254 in E. coli host strain NM514. A phage lysate ofthis material has been deposited as at the American Type CultureCollection under ATCC Accession No. 75293, as described above. DNAencoding α_(1A) may also be identified by screening with a probeprepared from DNA that has SEQ ID No. 21:5° CTCAGTACCATCTCTGATACCAGCCCCA 3′.

α_(1A) splice variants have been obtained. The sequences of two α_(1A)splice variants, α_(1a-1) and α_(1a-2) are set forth in SEQ. ID Nos. 22and 23. Other splice variants may be obtained by screening a humanlibrary as described above or using all or a portion of the sequencesset forth in SEQ ID Nos. 22 and 23.

Identification and Isolation of DNA Encoding the α_(1B) Human CalciumChannel Subunit

DNA encoding the α_(1B) subunit was isolated by screening a human basalganglia cDNA library with fragments of the rabbit skeletal musclecalcium channel α₁ subunit-encoding cDNA. A portion of one of thepositive clones was used to screen an IMR32 cell cDNA library. Clonesthat hybridized to the basal ganglia DNA probe were used to furtherscreen an IMR32 cell cDNA library to identify overlapping clones that inturn were used to screen a human hippocampus cDNA library. In this way,a sufficient series of clones to span nearly the entire length of thenucleotide sequence encoding the human α_(1B) subunit was obtained. PCRamplification of specific regions of the IMR32 cell α_(1B) mRNA yieldedadditional segments of the α_(1B) coding sequence.

A full-length α_(1B) DNA clone was constructed by ligating portions ofthe partial cDNA clones as described in Example II.C. SEQ ID Nos. 7 and8 show the nucleotide sequences of DNA clones encoding the α_(1B)subunit as well as the deduced amino acid sequences. The α_(1B) subunitencoded by SEQ ID No. 7 is referred to as the α_(1B-1) subunit todistinguish it from another α_(1B) subunit, α_(1b-2), encoded by thenucleotide sequence shown as SEQ ID No. 8, which is derived fromalternative splicing of the α_(1B) subunit transcript.

PCR amplification of IMR32 cell mRNA using oligonucleotide primersdesigned according to nucleotide sequences within the α_(1B-1)-encodingDNA has identified variants of the α_(1B) transcript that appear to besplice variants because they contain divergent coding sequences.

Identification and Isolation of DNA Encoding the α_(1C) Human CalciumChannel Subunit

Numerous α_(1C)-specific DNA clones were isolated. Characterization ofthe sequence revealed the α_(1C) coding sequence, the α_(1C) initiationof translation sequence, and an alternatively spliced region of α_(1C).Alternatively spliced variants of the α_(1C) subunit have beenidentified. SEQ ID No. 3 sets forth DNA encoding an α_(1C) subunit. TheDNA sequences set forth in SEQ ID No. 4 and No. 5 encode two possibleamino terminal ends of the tic protein. SEQ ID No. 6 encodes analternative exon for the IV S3 transmembrane domain.

The isolation and identification of DNA clones encoding portions of theα_(1C) subunit is described in detail in Example II.

Identification and Isolation of DNA Encoding the α_(1E) Human CalciumChannel Subunit

DNA encoding α_(1E) human calcium channel subunits have been isolatedfrom an oligo dT-primed human hippocampus library. The resulting clones,which are splice variants, were designated α_(1E-1) and α_(1E-3). Thesubunit designated α_(1E-1) has the amino acid sequence set forth in SEQID No. 24, and a subunit designated α_(1E-3) has the amino acid sequenceset forth by SEQ. ID No. 24 with the fragment encoded by the DNA setforth in SEQ ID No. 25 inserted between nucleotides 2405 and 2406. Theresulting sequence of α_(1E-3) is set forth in SEQ ID No. 27.

The α_(1E) subunits propvided herein appear to participate in theformation of calcium channels that have properties of high-voltageactivated calcium channels and low-voltage activated channels. Thesechannels are rapidly inactivating compared to other highvoltage-activated calcium channels. In addition these channels exhibitpharmacological profiles that are similar to voltage-activated channels,but are also sensitive to DHPs and ω-Aga-IVA, which block certain highvoltage activated channels. Additional details regarding theelectrophysiology and pharmacology of channels containing α_(1E)subunits is provided in Example VII. F.

Identification and Isolation of DNA Encoding the Other α₁ Human CalciumChannel Subunit Types and Subtypes

DNA encoding other α₁ subunits has also been isolated. Additional suchsubunits may also be isolated and identified using the DNA providedherein as described for the α_(1B), α_(1C) and α_(1D) subunits or usingother methods known to those of skill in the art. In particular, the DNAprovided herein may be used to screen appropriate libraries to isolaterelated DNA. Full-length clones can be constructed using methods, suchas those described herein, and the resulting subunits characterized bycomparison of their sequences and electrophysiological andpharmacological properties with the subunits exemplified herein.

Identification and Isolation DNA Encoding β Human Calcium ChannelSubunits

DNA Encoding β₁

To isolate DNA encoding the β₁ subunit, a human hippocampus cDNA librarywas screened by hybridization to a DNA fragment encoding a rabbitskeletal muscle calcium channel β subunit. A hybridizing clone wasselected and was in turn used to isolate overlapping clones until theoverlapping clones encompassing DNA encoding the entire the humancalcium channel β subunit were isolated and sequenced.

Five alternatively spliced forms of the human calcium channel β₁ subunithave been identified and DNA encoding a number of forms have beenisolated. These forms are designated β₁₋₁, expressed in skeletal muscle,β₁₋₂, expressed in the CNS, β₁₋₃, also expressed in the in the CNS,β₁₋₄, expressed in aorta tissue and HEK 293 cells, and β₁₋₅, expressedin HEK 293 cells. A full-length DNA clone encoding the β₁₋₂ subunit hasbeen constructed. The subunits β₁₋₁, β₁₋₂, β₁₋₄ and β₁₋₅ have beenidentified by PCR analysis as alternatively spliced forms of the βsubunit.

The alternatively spliced variants were identified by comparison ofamino acid sequences encoded by the human neuronal and rabbit skeletalmuscle calcium channel β subunit-encoding DNA. This comparison revealeda 45-amino acid deletion in the human β subunit compared to the rabbit βsubunit. Using DNA from the region as a probe for DNA cloning, as wellas PCR analysis and DNA sequencing of this area of sequence divergence,alternatively spliced forms of the human calcium channel β subunittranscript were identified. For example, the sequence of DNA encodingone splice variant β₁₋₂ is set forth in SEQ ID No. 9. SEQ ID No. 10 setsforth the sequence of the β₁₋₃ subunit (nt 1-1851, including 3′untranslated sequence nt 1795-1851), which is another splice variant ofthe β subunit primary transcript. β₁₋₂ and β₁₋₃ are human neuronal βsubunits. DNA distinctive for a portion of a β subunit (β₁₋₄) of a humanaortic calcium channel and also human embryonic kidney (HEK) cells isset forth in SEQ ID No. 12 (nt 1-13 and 191-271). The sequence of DNAencoding a portion of a human calcium channel β subunit expressed inskeletal muscle (β₁₋₁) is shown in SEQ ID No. 12 (nt 1-13 and 35-271).The sequences of the β₁ splice variants designated ⊕₁₋₁, β₁₋₂, β₁₋₃,β₁₋₄ and β₁₋₅ are set forth in Sequence ID Nos. 32, 9, 10, 33 and 34,respectively.

DNA Encoding β₂

DNA encoding the β₂ splice variants has been obtained. These splicevariants include β_(2A)-β_(2F). Splice variants β_(2C)-β_(2F) includeall of sequence set forth in SEQ ID No. 26, except for the portion atthe 5′ end (up to nucleotide 182), which differs among splice variants.The sequence set forth in SEQ ID No. 26 encodes at least about 90% ofβ_(2D). Additional splice variants may be isolated using the methodsdescribed herein and oligonucleotides including all or portions of theDNA set forth in SEQ ID. No. 26 or as described in the Examples.

DNA Encoding β₃

DNA encoding the β₃ subunit and any splice variants thereof may beisolated by screening a library, as described above for the β₁ subunit,using DNA probes prepared according to SEQ ID Nos. 19, 20 or using allor a portion of the deposited β₃ clone plasmid β1.42 (ATCC Accession No.69048).

The E. coli host containing plasmid β1.42 that includes DNA encoding aβ₃ subunit has been deposited as ATCC Accession No. 69048 in theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under the terms of the Budapest Treaty on the InternationalRecognition of Deposits of Microorganisms for Purposes of PatentProcedure and the Regulations promulgated under this Treaty. Samples ofthe deposited material are and will be available to industrial propertyoffices and other persons legally entitled to receive them under theterms of said Treaty and Regulations and otherwise in compliance withthe patent laws and regulations of the United States of America and allother nations or international organizations in which this application,or an application claiming priority of this application, is filed or inwhich any patent granted on any such application is granted.

The β₃ encoding plasmid is designated β1.42. The plasmid contains a 2.5kb EcoRI fragment encoding β₃ inserted into vector pGem^(•)7zF(+) andhas been deposited in E. coli host strain DN5α. A partial DNA sequenceof the 5′ and 3′ ends of β₃ are set forth in SEQ ID Nos. 19 and 20,respectively.

Identification and Isolation DNA Encoding the α₂ Human Calcium ChannelSubunit

DNA encoding a human neuronal calcium channel α₂ subunit was isolated ina manner substantially similar to that used for isolating DNA encodingan α₁ subunit, except that a human genomic DNA library was probed underlow and high stringency conditions with a fragment of DNA encoding therabbit skeletal muscle calcium channel α₂ subunit. The fragment includednucleotides having a sequence corresponding to the nucleotide sequencebetween nucleotides 43 and 272 inclusive of rabbit back skeletal musclecalcium channel α₂ subunit cDNA as disclosed in PCT International PatentApplication Publication No. WO 89/09834, which corresponds to U.S.application Ser. No. 07/620,520, which is a continuation-in-part of U.S.Ser. No. 176,899, filed Apr. 4, 1988, which applications have beenincorporated herein by reference. Example IV describes the isolation ofDNA clones encoding α₂ subunits of a human calcium channel from a humanDNA library using genomic DNA and cDNA clones, identified byhybridization to the genomic DNA, as probes.

SEQ ID No. 11 shows the sequence of DNA encoding an α₂ subunit. Asdescribed in Example V, PCR analysis of RNA from human skeletal muscle,brain tissue and aorta using oligonucleotide primers specific for aregion of the human neuronal α₂ subunit cDNA that diverges from therabbit skeletal muscle calcium channel α₂ subunit cDNA identified splicevariants of the human calcium channel α₂ subunit transcript.

Identification and Isolation of DNA Encoding γ Human Calcium ChannelSubunits

DNA encoding a human neuronal calcium channel γ subunit has beenisolated as described in detail in Example VI. SEQ ID No. 14 shows thenucleotide sequence at the 3′-end of this DNA which includes a readingframe encoding a sequence of 43 amino acid residues.

Preparation of Recombinant Eukaryotic Cells Containing DNA EncodingHeterologous Calcium Channel Subunits

DNA encoding one or more of the calcium channel subunits or a portion ofa calcium channel subunit may be introduced into a host cell forexpression or replication of the DNA. Such DNA may be introduced usingmethods described in the following examples or using other procedureswell known to those skilled in the art. Incorporation of cloned DNA intoa suitable expression vector, transfection of eukaryotic cells with aplasmid vector or a combination of plasmid vectors, each encoding one ormore distinct genes or with linear DNA, and selection of transfectedcells are also well known in the art [see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press].

Cloned full-length DNA encoding any of the subunits of a human calciumchannel may be introduced into a plasmid vector for expression in aeukaryotic cell. Such DNA may be genomic DNA or cDNA. Host cells may betransfected with one or a combination of said plasmids, each of whichencodes at least one calcium channel subunit. Alternatively, host cellsmay be transfected with linear DNA using methods well known to those ofskill in the art.

While the DNA provided herein may be expressed in any eukaryotic cell,including yeast cells such as P. pastoris [see, e.g., Cregg et al.(1987) Bio/Technology 5:479], mammalian expression systems forexpression of the DNA encoding the human calcium channel subunitsprovided herein are preferred.

The heterologous DNA may be introduced by any method known to those ofskill in the art, such as transfection with a vector encoding theheterologous DNA. Particularly preferred vectors for transfection ofmammalian cells are the pSV2dhfr expression vectors, which contain theSV40 early promoter, mouse dhfr gene, SV40 polyadenylation and splicesites and sequences necessary for maintaining the vector in bacteria,cytomegalovirus (CMV) promoter-based vectors such as pCMV or pCDNA1, andMMTV promoter-based vectors. DNA encoding the human calcium channelsubunits has been inserted in the vector pCDNA1 at a positionimmediately following the CMV promoter.

Stably or transiently transfected mammalian cells may be prepared bymethods known in the art by transfecting cells with an expression vectorhaving a selectable marker gene such as the gene for thymidine kinase,dihydrofolate reductase, neomycin resistance or the like, and, fortransient transfection, growing the transfected cells under conditionsselective for cells expressing the marker gene. Functionalvoltage-dependent calcium channels have been produced in HEK 293 cellstransfected with a derivative of the vector pCDNA1 that contains DNAencoding a human calcium channel subunit.

The heterologous DNA may be maintained in the cell as an episomalelement or may be integrated into chromosomal DNA of the cell. Theresulting recombinant cells may then be cultured or subcultured (orpassaged, in the case of mammalian cells) from such a culture or asubculture thereof. Methods for transfection, injection and culturingrecombinant cells are known to the skilled artisan. Eukaryotic cells inwhich DNA or RNA may be introduced, include any cells that aretransfectable by such DNA or RNA or into which such DNA may be injected.Virtually any eukaryotic cell can serve as a vehicle for heterologousDNA. Preferred cells are those that can also express the DNA and RNA andmost preferred cells are those that can form recombinant or heterologouscalcium channels that include one or more subunits encoded by theheterologous DNA. Such cells may be identified empirically or selectedfrom among those known to be readily transfected or injected. Preferredcells for introducing DNA include those that can be transiently orstably transfected and include, but are not limited to cells ofmammalian origin, such as COS cells, mouse L cells, CHO cells, humanembryonic kidney cells, African green monkey cells and other such cellsknown to those of skill in the art, amphibian cells, such as Xenopuslaevis oöcytes, or those of yeast such as Saccharomyces cerevisiae orPichia pastoris. Preferred cells for expressing injected RNA transcriptsinclude Xenopus laevis oöcytes. Cells that are preferred fortransfection of DNA are those that can be readily and efficientlytransfected. Such cells are known to those of skill in the art or may beempirically identified. Preferred cells include DG44 cells and HEK 293cells, particularly HEK 293 cells that have been adapted for growth insuspension and that can be frozen in liquid nitrogen and then thawed andregrown. Such HEK 293 cells are described, for example in U.S. Pat. No.5,024,939 to Gorman [see, also Stillman et al. (1985) Mol. Cell.Biol.5:2051-2060].

The cells may be used as vehicles for replicating heterologous DNAintroduced therein or for expressing the heterologous DNA introducedtherein. In certain embodiments, the cells are used as vehicles forexpressing the heterologous DNA as a means to produce substantially purehuman calcium channel subunits or heterologous calcium channels. Hostcells containing the heterologous DNA may be cultured under conditionswhereby the calcium channels are expressed. The calcium channel subunitsmay be purified using protein purification methods known to those ofskill in the art. For example, antibodies, such as those providedherein, that specifically bind to one or more of the subunits may beused for affinity purification of the subunit or calcium channelscontaining the subunits.

Substantially pure subunits of a human calcium channel α₁ subunits of ahuman calcium channel, α₂ subunits of a human calcium channel, βsubunits of a human calcium channel and γ subunits of a human calciumchannel are provided. Substantially pure isolated calcium channels thatcontain at least one of the human calcium channel subunits are alsoprovided. Substantially pure calcium channels that contain a mixture ofone or more subunits encoded by the host cell and one or more subunitsencoded by heterologous DNA or RNA that has been introduced into thecell are also provided. Substantially pure subtype- or tissue-typespecific calcium channels are also provided.

In other embodiments, eukaryotic cells that contain heterologous DNAencoding at least one of an α₁ subunit of a human calcium channel, an α₂subunit of a human calcium channel, a αsubunit of a human calciumchannel and a γ subunit of a human calcium channel are provided. Inaccordance with one preferred embodiment, the heterologous DNA isexpressed in the eukaryotic cell and preferably encodes a human calciumchannel α₁ subunit.

Expression of Heterologous Calcium Channels: Electrophysiology andPharmacology

Electrophysiological methods for measuring calcium channel activity arekwown to those of skill in the art and are exemplified herein. Any suchmethods may be used in order to detect the formation of functionalcalcium channels and to characterize the kinetics and othercharacteristics of the resulting currents. Pharmacological studies maybe combined with the electrophysiological measurements in order tofurther characterize the calcium channels.

With respect to measurement of the activity of functional heterologouscalcium channels, preferably, endogenous ion channel activity and, ifdesired, heterologous channel activity of channels that do not containthe desired subunits, of a host cell can be inhibited to a significantextent by chemical, pharmacological and electrophysiological means,including the use of differential holding potential, to increase the S/Nratio of the measured heterologous calcium channel activity.

Thus, various combinations of subunits encoded by the DNA providedherein are introduced into eukaryotic cells. The resulting cells can beexamined to ascertain whether functional channels are expressed and todetermine the properties of the channels. In particularly preferredaspects, the eukaryotic cell which contains the heterologous DNAexpresses it and forms a recombinant functional calcium channelactivity. In more preferred aspects, the recombinant calcium channelactivity is readily detectable because it is a type that is absent fromthe untransfected host cell or is of a magnitude not exhibited in theuntransfected cell.

The eukaryotic cells can be transfected with various combinations of thesubunit subtypes provided herein. The resulting cells will provide auniform population of calcium channels for study of calcium channelactivity and for use in the drug screening assays provided herein.Experiments that have been performed have demonstrate the inadequacy ofprior classification schemes.

Preferred among transfected cells is a recombinant eukaryotic cell witha functional heterologous calcium channel. The recombinant cell can beproduced by introduction of and expression of heteroloqous DNA or RNAtranscripts encoding an α₁ subunit of a human calcium channel, morepreferably also expressing, a heterologous DNA encoding a β subunit of ahuman calcium channel and/or heterologous DNA encoding an α₂ subunit ofa human calcium channel. Especially preferred is the expression in sucha recombinant cell of each of the α₁, β and α₂ subunits encoded by suchheterologous DNA or RNA transcripts, and optionally expression ofheterologous DNA or an RNA transcript encoding a γ subunit of a humancalcium channel. The functional calcium channels may preferably includeat least an α₁ subunit and a P subunit of a human calcium channel.Eukaryotic cells expressing these two subunits and also cells expressingadditional subunits, have been prepared by transfection of DNA and byinjection of RNA transcripts. Such cells have exhibitedvoltage-dependent calcium channel activity attributable to calciumchannels that contain one or more of the heterologous human calciumchannel subunits. For example, eukaryotic cells expressing heterologouscalcium channels containing an α₂ subunit in addition to the α₁ subunitand a β subunit have been shown to exhibit increased calcium selectiveion flow across the cellular membrane in response to depolarization,indicating that the α₂ subunit may potentiate calcium channel function.

Eukaryotic cells which express heterologous calcium channels containingat least a human α₁ subunit, a human β subunit and a human α₂ subunitare preferred. Eukaryotic cells transformed with a compositioncontaining cDNA or an RNA transcript that encodes an α₁ subunit alone orin combination with a β and/or an α₂ subunit may be used to producecells that express functional calcium channels. Since recombinant cellsexpressing human calcium channels containing all of the of the humansubunits encoded by the heterologous cDNA or RNA are especiallypreferred, it is desirable to inject or transfect such host cells with asufficient concentration of the subunit-encoding nucleic acids to formcalcium channels that contain the human subunits encoded by heterologousDNA or RNA. The precise amounts and ratios of DNA or RNA encoding thesubunits may be empirically determined and optimized for a particularcombination of subunits, cells and assay conditions.

In particular, mammalian cells have been transiently and stablytranfected with DNA encoding one or more human calcium channel subunits.Such cells express heterologous calcium channels that exhibitpharmacological and electrophysiological properties characteristic thatcan be ascribed to human calcium channels. Such cells, however,represent homogeneous populations and the pharmacological andelectrophysiological data provides insights into human calcium channelactivity heretofore unattainable. For example, HEK cells that have beentransiently transfected with DNA encoding the α1E-1, α_(2b), and β₁₋₃subunits. The resulting cells transiently express these subunits, whichform a calcium channels that appear to exhibit properties of L-, N-, T-and P-type channels.

HEK cells that have been transfiently transfected with DNA encodingα_(1B-1) α_(2b), and β₁₋₂ express heterologous calcium channels thatexhibt sensitivity to ω-conotoxin and currents typical of N-typechannels. It has been found that alteration of the molar raios ofα_(1B-1), α_(2b) and β₁₋₂ introduced into the cells into to achieveequivalent mRNA levles significantly incresed the number of receptorsper cell, the current density, and affected the K_(d) for ω-conotoxin.

The electrophyiology of these channels produced from α_(1B-1) α_(2b),and β₁₋₂ was compared with channels produced by transiently transfectingHEK cells with DNA encoding α_(1B-1) α_(2b), and β₁₋₃. The channelsexhibited similar voltage dependence of activation, substantiallyidentical voltage dependence, similar kinetics of activation and tailcurrents that could be fit by a single exponential. The voltagedependence of the kinetics of inactivation was significantly differentat all voltages examined.

In certain embodiments, the eukaryotic cell with a heterologous calciumchannel is produced by introducing into the cell a first composition,which contains at least one RNA transcript that is translated in thecell into a subunit of a human calcium channel. In preferredembodiments, the subunits that are translated include an α₁ subunit of ahuman calcium channel. More preferably, the composition that isintroduced contains an RNA transcript which encodes an α₁ subunit of ahuman calcium channel and also contains (1) an RNA transcript whichencodes a β subunit of a human calcium channel and/or (2) an RNAtranscript which encodes an α₂ subunit of a human calcium channel.Especially preferred is the introduction of RNA encoding an α₁, a β andan α₂ human calcium channel subunit, and, optionally, a γ subunit of ahuman calcium channel.

Methods for in vitro transcription of a cloned DNA and injection of theresulting RNA into eukaryotic cells are well known in the art.Transcripts of any of the full-length DNA encoding any of the subunitsof a human calcium channel may be injected alone or in combination withother transcripts into eukaryotic cells for expression in the cells.Amphibian oöcytes are particularly preferred for expression of in vitrotranscripts of the human calcium channel subunit cDNA clones providedherein. Amphibian oöcytes that express functional heterologous calciumchannels have been produced by this method.

Assays

Assays for Identifying Compounds that Modulate Calcium Channel Activity

Among the uses for eukaryotic cells which recombinantly express one ormore subunits are assays for determining whether a test compound hascalcium channel agonist or antagonist activity. These eukaryotic cellsmay also be used to select from among known calcium channel agonists andantagonists those exhibiting a particular calcium channe subtypespecificity and to thereby select compounds that have potential asdisease- or tissue-specific therapeutic agents.

In vitro methods for identifying compounds, such as calcium channelagonist and antagonists, that modulate the activity of calcium channelsusing eukaryotic cells that express heterologous human calcium channelsare provided.

In particular, the assays use eukaryotic cells that express heterologoushuman calcium channel subunits encoded by heterologous DNA providedherein, for screening potential calcium channel agonists and antagonistswhich are specific for human calcium channels and particularly forscreening for compounds that are specific for particular human calciumchannel subtypes. Such assays may be used in conjunction with methods ofrational drug design to select among agonists and antagonists, whichdiffer slightly in structure, those particularly useful for modulatingthe activity of human calcium channels, and to design or selectcompounds that exhibit subtype- or tissue-specific calcium channelantagonist and agonist activities.

These assays should accurately predict the relative therapeutic efficacyof a compound for the treatment of certain disorders in humans. Inaddition, since subtype- and tissue-specific calcium channel subunitsare provided, cells with tissue-specific or subtype-specific recombinantcalcium channels may be prepared and used in assays for identificationof human calcium channel tissue- or subtype-specific drugs.

Desirably, the host cell for the expression of calcium channel subunitsdoes not produce endogenous calcium channel subunits of the type or inan amount that substantially interferes with the detection ofheterologous calcium channel subunits in ligand binding assays ordetection of heterologous calcium channel function, such as generationof calcium current, in functional assays. Also, the host cellspreferably should not produce endogenous calcium channels whichdetectably interact with compounds having, at physiologicalconcentrations (generally nanomolar or picomolar concentrations),affinity for calcium channels that contain one or all of the humancalcium channel subunits provided herein.

With respect to ligand binding assays for identifying a compound whichhas affinity for calcium channels, cells are employed which express,preferably, at least a heterologous α₁ subunit. Transfected eukaryoticcells which express at least an α₁ subunit may be used to determine theability of a test compound to specifically alter the activity of acalcium channel. Such ligand binding assays may be performed on intacttransfected cells or membranes prepared therefrom.

The capacity of a test compound to bind to or otherwise interact withmembranes that contain heterologous calcium channels or subunits thereofmay be determined by using any appropriate method, such as competitivebinding analysis, such as Scatchard plots, in which the binding capacityof such membranes is determined in the presence and absence of one ormore concentrations of a compound having known affinity for the calciumchannel. Where necessary, the results may be compared to a controlexperiment designed in accordance with methods known to those of skillin the art. For example, as a negative control, the results may becompared to those of assays of an identically treated membranepreparation from host cells which have not been transfected with one ormore subunit-encoding nucleic acids.

The assays involve contacting the cell membrane of a recombinanteukaryotic cell which expresses at least one subunit of a human calciumchannel, preferably at least an α₁ subunit of a human calcium channel,with a test compound and measuring the ability of the test compound tospecifically bind to the membrane or alter or modulate the activity of aheterologous calcium channel on the membrane.

In preferred embodiments, the assay uses a recombinant cell that has acalcium channel containing an α₁ subunit of a human calcium channel incombination with a β-subunit of a human calcium channel and/or an α₂subunit of a human calcium channel. Recombinant cells expressingheterologous calcium channels containing each of the α₁, β and α₂ humansubunits, and, optionally, a γ subunit of a human calcium channel areespecially preferred for use in such assays.

In certain embodiments, the assays for identifying compounds thatmodulate calcium channel activity are practiced by measuring the calciumchannel activity of a eukaryotic cell having a heterologous, functionalcalcium channel when such cell is exposed to a solution containing thetest compound and a calcium channel selective ion and comparing themeasured calcium channel activity to the calcium channel activity of thesame cell or a substantially identical control cell in a solution notcontaining the test compound. The cell is maintained in a solutionhaving a concentration of calcium channel selective ions sufficient toprovide an inward current when the channels open. Especially preferredfor use, is a recombinant cell expressing calcium channels that includeeach of the α₁, β and α₂ human subunits, and, optionally, a γ subunit ofa human calcium channel. Methods for practicing such assays are known tothose of skill in the art. For example, for similar methods applied withXenopus laevis oöcytes and acetylcholine receptors, see, Mishina et al.[(1985) Nature 313:364] and, with such oöcytes and sodium channels [see,Noda et al. (1986) Nature 322:826-828]. For similar studies which havebeen carried out with the acetylcholine receptor, see, e.g., Claudio etal. [(1987) Science 238:1688-1694].

Functional recombinant or heterologous calcium channels may beidentified by any method known to those of skill in the art. Forexample, electrophysiological procedures for measuring the currentacross an ion-selective membrane of a cell, which are well known, may beused. The amount and duration of the flow of calcium selective ionsthrough heterologous calcium channels of a recombinant cell containingDNA encoding one or more of the subunits provided herein has beenmeasured using electrophysiological recordings using a two electrode andthe whole-cell patch clamp techniques. In order to improve thesensitivity of the assays, known methods can be used to eliminate orreduce non-calcium currents and calcium currents resulting fromendogenous calcium channels, when measuring calcium currents throughrecombinant channels. For example, the DHP Bay K 8644 specificallyenhances L-type calcium channel function by increasing the duration ofthe open state of the channels [see, e.g., Hess, J. B., et al. (1984)Nature 311:538-544]. Prolonged opening of the channels results incalcium currents of increased magnitude and duration. Tail currents canbe observed upon repolarization of the cell membrane after activation ofion channels by a depolarizing voltage command. The opened channelsrequire a finite time to close or “deactivate” upon repolarization, andthe current that flows through the channels during this period isreferred to as a tail current. Because Bay K 8644 prolongs openingevents in calcium channels, it tends to prolong these tail currents andmake them more pronounced.

In practicing these assays, stably or transiently transfected cells orinjected cells that express voltage-dependent human calcium channelscontaining one or more of the subunits of a human calcium channeldesirably may be used in assays to identify agents, such as calciumchannel agonists and antagonists, that modulate calcium channelactivity. Functionally testing the activity of test compounds, includingcompounds having unknown activity, for calcium channel agonist orantagonist activity to determine if the test compound potentiates,inhibits or otherwise alters the flow of calcium through a human calciumchannel can be accomplished by (a) maintaining a eukaryotic cell whichis transfected or injected to express a heterologous functional calciumchannel capable of regulating the flow of calcium channel selective ionsinto the cell in a medium containing calcium channel selective ions (i)in the presence of and (ii) in the absence of a test compound; (b)maintaining the cell under conditions such that the heterologous calciumchannels are substantially closed and endogenous calcium channels of thecell are substantially inhibited (c) depolarizing the membrane of thecell maintained in step (b) to an extent and for an amount of timesufficient to cause (preferably, substantially only) the heterologouscalcium channels to become permeable to the calcium channel selectiveions; and (d) comparing the amount and duration of current flow into thecell in the presence of the test compound to that of the current flowinto the cell, or a substantially similar cell, in the absence of thetest compound.

The assays thus use cells, provided herein, that express heterologousfunctional calcium channels and measure functionally, such aselectrophysiologically, the ability of a test compound to potentiate,antagonize or otherwise modulate the magnitude and duration of the flowof calcium channel selective ions, such as Ca⁺⁺ or Ba⁺⁺, through theheterologous functional channel. The amount of current which flowsthrough the recombinant calcium channels of a cell may be determineddirectly, such as electrophysiologically, or by monitoring anindependent reaction which occurs intracellularly and which is directlyinfluenced in a calcium (or other) ion dependent manner.

Any method for assessing the activity of a calcium channel may be usedin conjunction with the cells and assays provided herein. For example,in one embodiment of the method for testing a compound for its abilityto modulate calcium channel activity, the amount of current is measuredby its modulation of a reaction which is sensitive to calcium channelselective ions and uses a eukaryotic cell which expresses a heterologouscalcium channel and also contains a transcriptional control elementoperatively linked for expression to a structural gene that encodes anindicator protein. The transcriptional control element used fortranscription of the indicator gene is responsive in the cell to acalcium channel selective ion, such as Ca²⁺ and Ba⁺. The details of suchtranscriptional based assays are described in commonly owned PCTInternational Patent Application No. PCT/US91/5625, filed Aug. 7, 1991,which claims priority to copending commonly owned U.S. application Ser.No. 07/563,751, filed Aug. 7, 1990, the contents of which applicationsare herein incorporated by reference thereto.

Assays for Diagnosis of LES

LES is an autoimmune disease characterized by an insufficient release ofacetylcholine from motor nerve terminals which normally are responsiveto nerve impulses. Immunoglobulins (IgG) from LES patients blockindividual voltage-dependent calcium channels and thus inhibit calciumchannel activity [Kim and Neher, Science 239:405-408 (1988)]. Adiagnostic assay for Lambert Eaton Syndrome (LES) is provided herein.The diagnostic assay for LES relies on the immunological reactivity ofLES IgG with the human calcium channels or particular subunits alone orin combination or expressed on the surface of recombinant cells. Forexample, such an assay may be based on immunoprecipitation of LES IgG bythe human calcium channel subunits and cells that express such subunitsprovided herein.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example I

Preparation of Libraries Used for Isolation of DNA Encoding HumanNeuronal Voltage-dependent Calcium Channel Subunits

A. RNA Isolation

1. IMR32 Cells

IMR32 cells were obtained from the American Type Culture Collection(ATCC Accession No. CCL127, Rockville, Md.) and grown in DMEM, 10% fetalbovine serum, 1% penicillin/streptomycin (GIBCO, Grand Island, N.Y.)plus 1.0 mM dibutyryl cAMP (dbcAMP) for ten days. Total RNA was isolatedfrom the cells according to the procedure described by H. C. Birnboim[(1988) Nucleic Acids Research 16:1487-1497]. Poly(A⁺) RNA was selectedaccording to standard procedures [see, e.g., Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress; pg. 7.26-7.29].

2. Human Thalamus Tissue

Human thalamus tissue (2.34 g), obtained from the National NeurologicalResearch Bank, Los Angeles, Calif., that had been stored frozen at −70°C. was pulverized using a mortar and pestle in the presence of liquidnitrogen and the cells were lysed in 12 ml of lysis buffer (5 Mguanidinium isothiocyanate, 50 mM TRIS, pH 7.4, 10 mM EDTA, 5%β-mercaptoethanol). Lysis buffer was added to the lysate to yield afinal volume of 17 ml. N-laurylsarcosine and CsCl were added to themixture to yield final concentrations of 4% and 0.01 g/ml, respectively,in a final volume of 18 ml.

The sample was centrifuged at 9,000 rpm in a Sorvall SS34 rotor for 10min at room temperature to remove the insoluble material as a pellet.The supernatant was divided into two equal portions and each was layeredonto a 2-ml cushion of a solution of 5.7 M CsCl, 0.1 M EDTA contained inseparate centrifuge tubes to yield approximately 9 ml per tube. Thesamples were centrifuged in an SW41 rotor at 37,000 rpm for 24 h at 20°C.

After centrifugation, each RNA pellet was resuspended in 3 ml ETS (10 mMTRIS, pH 7.4, 10 mM EDTA, 0.2% SDS) and combined into a single tube. TheRNA was precipitated with 0.25 M NaCl and two volumes of 95% ethanol.

The precipitate was collected by centrifugation and resuspended in 4 mlPK buffer (0.05 M TRIS, pH 8.4, 0.14 M NaCl, 0.01 M EDTA, 1% SDS).Proteinase K was added to the sample to a final concentration of 200μg/ml. The sample was incubated at 22° C. for 1 h, followed byextraction with an equal volume of phenol:chloroform:isoamylalcohol(50:48:2) two times, followed by one extraction with an equal volume ofchloroform: isoamylalcohol (24:1). The RNA was precipitated with ethanoland NaCl. The precipitate was resuspended in 400 μl of ETS buffer. Theyield of total RNA was approximately 1.0 mg. Poly A⁺ RNA (30 μg) wasisolated from the total RNA according to standard methods as stated inExample I.A.1.

B. Library Construction

Double-stranded cDNA was synthesized according to standard methods [see,e.g., Sambrook et al. (1989) IN: Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press, Chapter 8]. Each library wasprepared in substantially the same manner except for differences in: 1)the oligonucleotide used to prime the first strand cDNA synthesis, 2)the adapters that were attached to the double-stranded cDNA, 3) themethod used to remove the free or unused adapters, and 4) the size ofthe fractionated cDNA ligated into the λ phage vector.

1. IMR32 cDNA library #1

Single-stranded cDNA was synthesized using IMR32 poly(A⁺) RNA (ExampleI.A.1.) as a template and was primed using oligo (dT)₁₂₋₁₈(Collaborative Research Inc., Bedford, Mass.). The single-stranded cDNAwas converted to double-stranded cDNA and the yield was approximately 2μg. EcoI adapters:5′-AATTCGGTACGTACACTCGAGC-3′=22-mer  (SEQ ID No.15)3′-GCCATGCATGTGAGCTCG-5′=18-mer  (SEQ ID No.16)also containing SnaBI and XhoI restriction sites were then added to thedouble-stranded cDNA according to the following procedure.a. Phosphorylation of 18-mer

The 18-mer was phosphorylated using standard methods [see, e.g.,Sambrook et al. (1989) IN: Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Chapter 8] by combining in a 10 μl totalvolume the 18-mer (225 pmoles) with [³²P]γ-ATP (7000 Ci/mmole; 1.0 μl)and kinase (2 U) and incubating at 37° C. for 15 minutes. Afterincubation, 1 μl 10 mM ATP and an additional 2 U of kinase were addedand incubated at 37° C. for 15 minutes.

Kinase was then inactivated by boiling for 10 minutes.

b. Hybridization of 22-mer

The 22-mer was hybridized to the phosphorylated 18-mer by addition of225 pmoles of the 22-mer (plus water to bring volume to 15 μl), andincubation at 65° C. for 5 minutes. The reaction was then allowed toslow cool to room temperature.

The adapters were thus present at a concentration of 15 pmoles/μl, andwere ready for cDNA-adapter ligation.

c. Ligation of Adapters to cDNA

After the EcoRI, SnaBI, XhoI adapters were ligated to thedouble-stranded cDNA using a standard protocol [see, e.g., Sambrook etal. (1989) IN: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Chapter 8], the ligase was inactivated byheating the mixture to 72° C. for 15 minutes. The following reagentswere added to the cDNA ligation reaction and heated at 37° C. for 30minutes: cDNA ligation reaction (20 μl), water (24 μl), 10×kinase buffer(3 μl), 10 mM ATP (1 μl) and kinase (2 μl of 2 U/μl). The reaction wasstopped by the addition of 2 μl 0.5M EDTA, followed by onephenol/chloroform extraction and one chloroform extraction.

d. Size Selection and Packaging of cDNA

The double-stranded cDNA with the EcoRI, SnaBI, XhoI adapters ligatedwas purified away from the free or unligated adapters using a 5 mlSepharose CL-4B column (Sigma, St. Louis, Mo.). 100 μl fractions werecollected and those containing the cDNA, determined by monitoring theradioactivity, were pooled, ethanol precipitated, resuspended in TEbuffer and loaded onto a 1% agarose gel. After the electrophoresis, thegel was stained with ethidium bromide and the 1 to 3 kb fraction was cutfrom the gel. The cDNA embedded in the agarose was eluted using the“Geneluter Electroelution System” (Invitrogen, San Diego, Calif.). Theeluted cDNA was collected by ethanol precipitation and resuspended in TEbuffer at 0.10 pmol/μl. The cDNA was ligated to 1 μg of EcoRI digested,dephosphorylated λgt11 in a 5 μl reaction volume at a 2- to 4-fold molarexcess ratio of cDNA over the λgt11 vector. The ligated λgt11 containingthe cDNA insert was packaged into λ phage virions in vitro using theGigapack (Stratagene, La Jolla, Calif.) kit. The packaged phage wereplated on an E. coli Y1088 bacterial lawn in preparation for screening.

2. IMR32 CDNA Library #2

This library was prepared as described (Example I.B.1.) with theexception that 3 to 9 kb cDNA fragments were ligated into the λgt11phage vector rather than the 1 to 3 kb fragments.

3. IMR32 cDNA Library #3

IMR32 cell poly(A⁺) RNA (Example I.A.1.) was used as a template tosynthesize single-stranded cDNA. The primers for the first strand cDNAsynthesis were random primers (hexadeoxy-nucleotides [pd(N)₆] Cat#5020-1, Clontech, Palo Alto, Calif.). The double-stranded cDNA wassynthesized, EcoRI, SnaBI, XhoI adapters were added to the cDNA, theunligated adapters were removed, and the double-stranded cDNA with theligated adapters was fractionated on an agarose gel, as described inExample I.B.1. The cDNA fraction greater than 1.8 kb was eluted from theagarose, ligated into λgt11, packaged, and plated into a bacterial lawnof Y1088 (as described in Example I.B.1.).

4. IMR32 cDNA Library #4

IMR32 cell poly(A⁺) RNA (Example I.A.1.) was used as a template tosynthesize single-stranded cDNA. The primers for the first strand cDNAsynthesis were oligonucleotides: 89-365a specific for the α_(1D) (VDCCIII) type α₁-subunit (see Example II.A.) coding sequence (thecomplementary sequence of nt 2927 to 2956, SEQ ID No. 1), 89-495specific for the α_(1C) (VDCC II) type α₁-subunit (see Example II.B.)coding sequence (the complementary sequence of nt 852 to 873, SEQ ID No.3), and 90-12 specific for the α_(1C)-subunit coding sequence (thecomplementary sequence of nt 2496 to 2520, SEQ ID No. 3). The cDNAlibrary was then constructed as described (Example I.B.3), except thatthe cDNA size-fraction greater than 1.5 kb was eluted from the agaroserather than the greater than 1.8 kb fraction.

5. IMR32 cDNA Library #5

The cDNA library was constructed as described (Example I.B.3.) with theexception that the size-fraction greater than 1.2 kb was eluted from theagarose rather than the greater than 1.8 kb fraction.

6. Human Thalamus cDNA Library #6

Human thalamus poly (A⁺) RNA (Example I.A.2.) was used as a template tosynthesize single-stranded cDNA. Oligo (dT) was used to prime the firststrand synthesis (Example I.B.1.). The double-stranded cDNA wassynthesized (Example I.B.1.) and EcoRI, KpnI, NcoI adapters of thefollowing sequence:5′ CCATGGTACCTTCGTTGACG 3′=20-mer  (SEQ ID NO. 17)3′ GGTACCATGGAAGCAACTGCTTAA 5′=24-mer  (SEQ ID NO. 18)were ligated to the double-stranded cDNA as described (Example I.B.1.)with the 20-mer replacing the 18-mer and the 24-mer replacing the22-mer. The unligated adapters were removed by passing the cDNA-adaptermixture through a 1 ml Bio Gel A-50 (Bio-Rad Laboratories, Richmond,Calif.) column. Fractions (30 μl) were collected and 1 μl of eachfraction in the first peak of radioactivity was electrophoresed on a 1%agarose gel. After electrophoresis, the gel was dried on a vacuum geldrier and exposed to x-ray film. The fractions containing cDNA fragmentsgreater than 600 bp were pooled, ethanol precipitated, and ligated intoλgt11 (Example I.B.1.). The construction of the cDNA library wascompleted as described (Example I.B.1.).C. Hybridization and Washing Conditions

Hybridization of radiolabelled nucleic acids to immobilized DNA for thepurpose of screening cDNA libraries, DNA Southern transfers, or northerntransfers was routinely performed in standard hybridization conditions[hybridization: 50% deionized formamide, 200 μg/ml sonicated herringsperm DNA (Cat #223646, Boehringer Mannheim Biochemicals, Indianapolis,Ind.), 5×SSPE, 5×Denhardt's, 42° C.; wash :0.2×SSPE, 0.1% SDS, 65° C.].The recipes for SSPE and Denhardt's and the preparation of deionizedformamide are described, for example, in Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Chapter 8). In some hybridizations, lower stringency conditionswere used in that 10% deionized formamide replaced 50% deionizedformamide described for the standard hybridization conditions.

The washing conditions for removing the non-specific probe from thefilters was either high, medium, or low stringency as described below:

-   -   1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.    -   2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.    -   3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.        It is understood that equivalent stringencies may be achieved        using alternative buffers, salts and temperatures.

Example II

Isolation of DNA Encoding the Human Neuronal Calcium Channel α₁ Subunit

A. Isolation of DNA Encoding the α_(1D) Subunit

1. Reference List of Partial α_(1D) cDNA Clones

Numerous α_(1D)-specific cDNA clones were isolated in order tocharacterize the complete α_(1D) coding sequence plus portions of the 5′and 3′ untranslated sequences. SEQ ID No. 1 shows the complete α_(1D)DNA coding sequence, plus 510 nucleotides of α_(1D) 5′ untranslatedsequence ending in the guanidine nucleotide adjacent to the adeninenucleotide of the proposed initiation of translation as well as 642nucleotides of 3′ untranslated sequence. Also shown in SEQ ID No. 1 isthe deduced amino acid sequence. A list of partial cDNA clones used tocharacterize the α_(1D) sequence and the nucleotide position of eachclone relative to the full-length α_(1D) cDNA sequence, which is setforth in SEQ ID No. 1, is shown below. The isolation andcharacterization of these clones are described below (Example II.A.2.).

IMR32 1.144 nt 1 to 510 of SEQ ID No. 1 5′ untranslated sequence, nt 511to 2431, SEQ ID No. 1 IMR32* 1.136 nt 1627 to 2988, SEQ ID No. 1 nt 1 to104 of SEQ ID No. 2 additional exon, IMR32@ 1.80 nt 2083 to 6468, SEQ IDNo. 1 IMR32# 1.36 nt 2857 to 4281, SEQ ID No. 1 IMR32 1.163 nt 5200 to7635, SEQ ID No. 1 *5′ of nt 1627, IMR32 1.136 encodes an intron and anadditional exon described in Example II.A.2.d. @IMR32 1.80 contains twodeletions, nt 2984 to 3131 and nt 5303 to 5349 (SEQ ID No. 1). The 148nt deletion (nt 2984 to 3131) was corrected by performing a polymerasechain reaction described in Example II.A.3.b. #IMR32 1.36 contains a 132nt deletion (nt 3081 to 3212).2. Isolation and Characterization of Individual Clones Listed in ExampleII.A.1.a. IMR32 1.36

Two million recombinants of the IMR32 cDNA library #1 (Example I.B.1.)were screened in duplicate at a density of approximately 200,000 plaquesper 150 mm plate using a mixture of radiolabelled fragments of thecoding region of the rabbit skeletal muscle calcium channel α₁ cDNA [forthe sequence of the rabbit skeletal muscle calcium channel α₁ subunitcDNA, see, Tanabe et al. (1987). Nature 328:313-318]:

Fragment Nucleotides KpnI-EcoRI  −78 to 1006 EcoRI-XboI 1006 to 2653ApaI-ApaI 3093 to 4182 BglII-SacI 4487 to 5310The hybridization was performed using low stringency hybridizationconditions (Example I.C.) and the filters were washed under lowstringency (Example I.C.). Only one α_(1D)-specific recombinant (IMR321.36) of the 2×10⁶ screened was identified. IMR32 1.36 was plaquepurified by standard methods (J. Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press,Chapter 8) subcloned into pGEM3 (Promega, Madison, Wis.) andcharacterized by DNA sequencing.b. IMR32 1.80

Approximately 1×106 recombinants of the IMR32 cDNA library #2 (ExampleI.B.2.) were screened in duplicate at a density of approximately 100,000plaques per 150 mm plate using the IMR32 1.36 cDNA fragment (ExampleII.A.1) as a probe. Standard hybridization conditions were used, and thefilters were washed under high stringency (Example I.C.). Three positiveplaques were identified one of which was IMR32 1.80. IMR32 1.80 wasplaque purified by standard methods, restriction mapped, subcloned, andcharacterized by DNA sequencing.

c. IMR32 1.144

Approximately 1×10⁶ recombinants of the IMR32 cDNA library #3 (ExampleI.B.3) were screened with the EcoRI-PvuII fragment (nt 2083 to 2518, SEQID No. 1) of IMR32 1.80. The hybridization was performed using standardhybridization conditions (Example I.C.) and the filters were washedunder high stringency (Example I.C.). Three positive plaques wereidentified one of which was IMR32 1.144. IMR32 1.144 was plaquepurified, restriction mapped, and the cDNA insert was subcloned intopGEM7Z (Promega, Madison, Wis.) and characterized by DNA sequencing.This characterization revealed that IMR32 1.144 has a series of ATGcodons encoding seven possible initiating methionines (nt 511 to 531,SEQ ID No. 1). PCR analysis, and DNA sequencing of cloned PCR productsencoding these seven ATG codons confirmed that this sequence is presentin the α_(1D) transcript expressed in dbcAMP-induced IMR32 cells.

d. IMR32 1.136

Approximately 1×10⁶ recombinants of the IMR32 cDNA library #4 (ExampleI.B.4) were screened with the EcoRI-PvuII fragment (nt 2083 to 2518, SEQID No. 1) of IMR32 1.80 (Example II.A.1.). The hybridization wasperformed using standard hybridization conditions (Example I.C.) and thefilters were washed under high stringency (Example I.C.). Six positiveplaques were identified one of which was IMR32 1.136. IMR32 1.136 wasplaque purified, restriction mapped, and the cDNA insert was subclonedinto a standard plasmid vector, pSP72 (Promega, Madison, Wis.), andcharacterized by DNA sequencing. This characterization revealed thatIMR32 1.136 encodes an incompletely spliced α_(1D) transcript. The clonecontains nucleotides 1627 to 2988 of SEQ ID No. 1 preceded by anapproximate 640 bp intron. This intron is then preceded by a 104 nt exon(SEQ ID No. 2) which is an alternative exon encoding the IS6transmembrane domain [see, e.g., Tanabe et al. (1987) Nature 328:313-318for a description of the IS1 to IVS6 transmembrane terminology] of theα_(1D) subunit and can replace nt 1627 to 1730, SEQ ID No. 1, to producea completely spliced α_(1D) transcript.

e. IMR32 1.163

Approximately 1×106 recombinants of the IMR32 cDNA library #3 (ExampleI.B.3.) were screened with the NcoI-XhoI fragment of IMR32 1.80 (ExampleII.A.1.) containing nt 5811 to 6468 (SEQ ID No. 1). The hybridizationwas performed using standard hybridization conditions (Example I.C.) andthe filters were washed under high stringency (Example I.C.). Threepositive plaques were identified one of which was IMR32 1.163. IMR321.163 was plaque purified, restriction mapped, and the cDNA insert wassubcloned into a standard plasmid vector, pSP72 (Promega, Madison,Wis.), and characterized by DNA sequencing. This characterizationrevealed that IMR32 1.163 contains the α_(1D) termination codon, nt 6994to 6996 (SEQ ID No. 1).

3. Construction of a Full-length α_(1D) cDNA [pVDCCIII(A)]

α_(1D) cDNA clones IMR32 1.144, IMR32 1.136, IMR32 1.80, and IMR32 1.163(Example II.A.2.) overlap and include the entire α_(1D) coding sequence,nt 511 to 6993 (SEQ ID No. 1), with the exception of a 148 bp deletion,nt 2984 to 3131 (SEQ ID No. 1). Portions of these partial cDNA cloneswere ligated to generate a full-length α_(1D) cDNA in a eukaryoticexpression vector. The resulting vector was called pVDCCIII(A). Theconstruction of PVDCCIII(A) was performed in four steps described indetail below: (1) the construction of pVDCCIII/5′ using portions ofIMR32 1.144, IMR32 1.136, and IMR32 1.80, (2) the construction ofpVDCCIII/5′.3 that corrects the 148 nt deletion in the IMR32 1.80portion of pVDCCIII/5′, (3) the construction of pVDCCIII/3′.1 usingportions of IMR32 1.80 and IMR32 1.163, and (4) the ligation of aportion of the pVDCCIII/5′.3 insert, the insert of pVDCCIII/3′.1, andpcDNA1 (Invitrogen, San Diego, Calif.) to form pVDCCIII(A). The vectorpcDNA1 is a eukaryotic expression vector containing a cytomegalovirus(CMV) promoter which is a constitutive promoter recognized by mammalianhost cell RNA polymerase II.

Each of the DNA fragments used in preparing the full-length constructwas purified by electrophoresis through an agarose gel onto DE81 filterpaper (Whatman, Clifton, N.J.) and elution from the filter paper using1.0 M NaCl, 10 mM TRIS, pH 8.0, 1 mM EDTA. The ligations typically wereperformed in a 10 μl reaction volume with an equal molar ratio of insertfragment and a two-fold molar excess of the total insert relative to thevector. The amount of DNA used was normally about 50 ng to 100 ng.

a. pVDCCIII/5′

To construct pVDCCIII/5′, IMR32 1.144 (Example II.A.2.c.) was digestedwith XhoI and EcoRI and the fragment containing the vector (pGEM7Z),α_(1D) nt 1 to 510 (SEQ ID No. 1), and α_(1D) nt 511 to 1732 (SEQ IDNo. 1) was isolated by gel electrophoresis. The EcoRI-ApaI fragment ofIMR32 1.136 (Example II.A.2.d.) nucleotides 1733 to 2671 (SEQ ID No. 1)was isolated, and the ApaI-HindIII fragment of IMR32 1.80 (ExampleII.A.2.b.), nucleotides 2672 to 4492 (SEQ ID No. 1) was isolated. Thethree DNA clones were ligated to form pVDCCIII/5′ containing nt 1 to 510(5′ untranslated sequence; SEQ ID No. 1) and nt 511 to 4492 (SEQ ID No.1).

b. pVDCCIII/5′.3

Comparison of the IMR32 1.36 and IMR32 1.80 DNA sequences revealed thatthese two cDNA clones differ through the α_(1D) coding sequence,nucleotides 2984 to 3212. PCR analysis of IMR32 1.80 and dbcAMP-induced(1.0 mM, 10 days) IMR32 cytoplasmic RNA (isolated according to Ausubel,F. M. et al. (Eds) (1988) Current Protocols in Molecular Biology, JohnWiley and Sons, New York) revealed that IMR32 1.80 had a 148 ntdeletion, nt 2984 to 3131 (SEQ ID No. 1), and that IMR32 1.36 had a 132nt deletion, nt 3081 to 3212. To perform the PCR analysis, amplificationwas primed with α_(1D)-specific oligonucleotides 112 (nt 2548 to 2572,SEQ ID No. 1) and 311 (the complementary sequence of nt 3928 to 3957,SEQ ID No. 1). These products were then reamplified usingα_(1D)-specific oligonucleotides 310 (nt 2583 to 2608 SEQ ID No. 1) and312 (the complementary sequence of nt 3883 to 3909). This reamplifiedproduct, which contains AccI and BGlII restriction sites, was digestedwith AccI and BGlII and the AccI-BGlII fragment, nt 2765 to 3890 (SEQ IDNo. 1) was cloned into AccI-BGlII digested pVDCCIII/5′ to replace theAccI-BGlII pVDCCIII/5′ fragment that had the deletion. This newconstruct was named pVDCCIII/5′.3. DNA sequence determination ofpVDCCIII/5′.3 through the amplified region confirmed the 148 nt deletionin IMR32 1.80.

c. pVDCCIII/3′.1

To construct pVDCCIII/3′.1, the cDNA insert of IMR32 1.163 (ExampleII.A.2.e.) was subcloned into pBluescript II (Stratagene, La Jolla,Calif.) as an XhoI fragment. The XhoI sites on the cDNA fragment werefurnished by the adapters used to construct the cDNA library (ExampleI.B.3.). The insert was oriented such that the translational orientationof the insert of IMR32 1.163 was opposite to that of the lacZ genepresent in the plasmid, as confirmed by analysis of restriction enzymedigests of the resulting plasmid. This was done to preclude thepossibility of expression of α_(1D) sequences in DH5α cells transformedwith this plasmid due to fusion with the lacZ gene. This plasmid wasthen digested with HindIII and BGlII and the HindIII-BGlII fragment (theHindIII site comes from the vector and the BGlII site is at nt 6220, SEQID No. 1) was eliminated, thus deleting nt 5200 to 6220 (SEQ ID No. 1)of the IMR32 1.163 clone and removing this sequence from the remainderof the plasmid which contained the 3′ BGlII-XhoI fragment, nt 6221 to7635 (SEQ ID No. 1). pVDCCIII/3′.1 was then made by splicing togetherthe HindIII-PvuII fragment from IMR32 1.80 (nucleotides 4493-5296, SEQID No. 1), the PvuII-BGlII fragment of IMR32 1.163 (nucleotides 5294 to6220, SEQ ID No. 1) and the HindIII-BGlII-digested pBluescript plasmidcontaining the 3′ BGlII/XhoI IMR32 1.163 fragment (nt 6221 to 7635, SEQID No. 1).

d. pVDCCIII(A): The Full-length α_(1D) Construct

To construct pVDCCIII(A), the DraI-HindIII fragment (5′ untranslatedsequence nt 330 to 510, SEQ ID No. 1 and coding sequence nt 511 to 4492,SEQ ID No. 1) of pVDCCIII/5′.3 (Example II.A.3.b.) was isolated; theHindIII-XhoI fragment of pVDCCIII/3′.1 (containing nt 4493 to 7635, SEQID No. 1, plus the XhoI site of the adapter) (Example II.A.3.c.) wasisolated; and the plasmid vector, pcDNA1, was digested with EcoRV andXhoI and isolated on an agarose gel. The three DNA fragments wereligated and MC1061-P3 (Invitrogen, San Diego, Calif.) was transformed.Isolated clones were analyzed by restriction mapping and DNA sequencingand PVDCCIII (A) was identified which had the fragments correctlyligated together: DraI-HindIII, HindIII-XhoI, XhoI-EcoRV with theblunt-end DraI and EcoRV site ligating together to form the circularplasmid.

The amino-terminus of the α_(1D) subunit is encoded by the sevenconsecutive 5′ methionine codons (nt 511 to 531, SEQ ID No. 1). This 5′portion plus nt 532 to 537, encoding two lysine residues, were deletedfrom pVDCCIII(A) and replaced with an efficient ribosomal binding site(5′-ACCACC-3′) to form pVDCCIII.RBS(A). Expression experiments in whichtranscripts of this construct were injected into Xenopus laevis oöcytesdid not result in an enhancement in the recombinant voltage-dependentcalcium channel expression level relative to the level of expression inoöcytes injected with transcripts of pVDCCIII(A).

B. Isolation of DNA Encoding the α_(1C) Subunit

1. Reference List of Partial α_(1C) cDNA Clones

Numerous α_(1C)-specific cDNA clones were isolated in order tocharacterize the α_(1C) coding sequence, the α_(1C) initiation oftranslation, and an alternatively spliced region of α_(1C). SEQ ID No. 3sets forth the characterized α_(1C) coding sequence (nt 1 to 5904) anddeduced amino acid sequence. SEQ ID No. 4 and No. 5 encode two possibleamino terminal ends of the α_(1C) protein. SEQ ID No. 6 encodes analternative exon for the IV S3 transmembrane domain. Shown below is alist of clones used to characterize the α_(1C) sequence and thenucleotide position of each clone relative to the characterized α_(1C)sequence (SEQ ID No. 3). The isolation and characterization of thesecDNA clones are described below (Example II.B.2).

IMR32 1.66 nt 1 to 916, SEQ ID No. 3 nt 1 to 132, SEQ ID No. 4 IMR321.157 nt 1 to 873, SEQ ID No. 3 nt 1 to 89, SEQ ID No. 5 IMR32 1.67 nt50 to 1717, SEQ ID No. 3 *IMR32 1.86 nt 1366 to 2583, SEQ ID No. 3@1.16G nt 758 to 867, SEQ ID No. 3 IMR32 1.37 nt 2804 to 5904, SEQ IDNo. 3 CNS 1.30 nt 2199 to 3903, SEQ ID No. 3 nt 1 to 84 of alternativeexon, SEQ ID No. 6 IMR32 1.38 nt 2448 to 4702, SEQ ID No. 3 nt 1 to 84of alternative exon, SEQ ID No. 6 *IMR32 1.86 has a 73 nt deletioncompared to the rabbit cardiac muscle calcium channel α₁ subunit cDNAsequence. @1.16G is an α_(1C) genomic clone.2. Isolation and Characterization of Clones Described in Example II.B.1.a. CNS 1.30

Approximately 1×10⁶ recombinants of the human thalamus cDNA library No.6 (Example I.B.6.) were screened with fragments of the rabbit skeletalmuscle calcium channel α₁ cDNA described in Example II.A.2.a. Thehybridization was performed using standard hybridization conditions(Example I.C.) and the filters were washed under low stringency (ExampleI.C.). Six positive plaques were identified, one of which was CNS 1.30.CNS 1.30 was plaque purified, restriction mapped, subcloned, andcharacterized by DNA sequencing. CNS 1.30 encodes α_(1C)-specificsequence nt 2199 to 3903 (SEQ ID No. 3) followed by nt 1 to 84 of one oftwo identified alternative α_(1C) exons (SEQ ID No. 6). 3′ of SEQ ID No.6, CNS 1.30 contains an intron and, thus, CNS 1.30 encodes a partiallyspliced α_(1C) transcript.

b. 1.16G

Approximately 1×10⁶ recombinants of a λEMBL3-based human genomic DNAlibrary (Cat # HL1006d Clontech Corp., Palo Alto, Calif.) were screenedusing a rabbit skeletal muscle cDNA fragment (nt −78 to 1006, ExampleII.A.2.a.). The hybridization was performed using standard hybridizationconditions (Example I.C.) and the filters were washed under lowstringency (Example I.C.). Fourteen positive plaques were identified,one of which was 1.16G. Clone 1.16G was plaque purified, restrictionmapped, subcloned, and portions were characterized by DNA sequencing.DNA sequencing revealed that 1.16G encodes α_(1C)-specific sequence asdescribed in Example II.B.1.

c. IMR32 1.66 and IMR32 1.67

Approximately 1×10⁶ recombinants of IMR32 cDNA library #5 (ExampleI.B.5.) were screened with a 151 bp KpnI-SacI fragment of 1.16G (ExampleII.B.2.b.) encoding α_(1C) sequence (nt 758 to 867, SEQ ID No. 3). Thehybridization was performed using standard hybridization conditions(Example I.C.). The filters were then washed in 0.5×SSPE at 65° C. Ofthe positive plaques, IMR32 1.66 and IMR32 1.67 were identified. Thehybridizing plaques were purified, restriction mapped, subcloned, andcharacterized by DNA sequencing. Two of these cDNA clones, IMR32 1.66and 1.67, encode α_(1C) subunits as described (Example II.B.1.). Inaddition, IMR32 1.66 encodes a partially spliced α_(1C) transcriptmarked by a GT splice donor dinucleotide beginning at the nucleotide 3′of nt 916 (SEQ ID No. 3). The intron sequence within 1.66 is 101 ntlong. IMR32 1.66 encodes the α_(1C) initiation of translation, nt 1 to 3(SEQ ID No. 3) and 132 nt of 5′ untranslated sequence (SEQ ID No. 4)precede the start codon in IMR32 1.66.

d. IMR32 1.37 and IMR32 1.38

Approximately 2×10⁶ recombinants of IMR32 cDNA library #1 (ExampleI.B.1.) were screened with the CNS 1.30 cDNA fragment (ExampleII.B.2.a.). The hybridization was performed using low stringencyhybridization conditions (Example I.C.) and the filters were washedunder low stringency (Example I.C.). Four positive plaques wereidentified, plaque purified, restriction mapped, subcloned, andcharacterized by DNA sequencing. Two of the clones, IMR32 1.37 and IMR321.38 encode α_(1C)-specific sequences as described in Example II.B.1.

DNA sequence comparison of IMR32 1.37 and IMR32 1.38 revealed that theα_(1C) transcript includes two exons that encode the IVS3 transmembranedomain. IMR32 1.37 has a single exon, nt 3904 to 3987 (SEQ ID No. 3) andIMR32 1.38 appears to be anomalously spliced to contain both exonsjuxtaposed, nt 3904 to 3987 (SEQ ID No. 3) followed by nt 1 to 84 (SEQID No. 6). The alternative splice of the α_(1C) transcript to containeither of the two exons encoding the IVS3 region was confirmed bycomparing the CNS 1.30 sequence to the IMR32 1.37 sequence. CNS 1.30contains nt 1 to 84 (SEQ ID No. 6) preceded by the identical sequencecontained in IMR32 1.37 for nt 2199 to 3903 (SEQ ID No. 3). As describedin Example II.B.2.a., an intron follows nt 1 to 84 (SEQ ID No. 6). Twoalternative exons have been spliced adjacent to nt 3903 (SEQ ID No. 3)represented by CNS 1.30 and IMR32 1.37.

e. IMR32 1.86

IMR32 cDNA library #1 (Example I.B.1.) was screened in duplicate usingoligonucleotide probes 90-9 (nt 1462 to 1491, SEQ ID No. 3) and 90-12(nt 2496 to 2520, SEQ ID No. 3). These oligonucleotide probes werechosen in order to isolate a clone that encodes the α_(1C) subunitbetween the 3′ end of IMR32 1.67 (nt 1717, SEQ ID No. 3) and the 5′ endof CNS 1.30 (nt 2199, SEQ ID No. 3). The hybridization conditions werestandard hybridization conditions (Example I.C.) with the exception thatthe 50% deionized formamide was reduced to 20%. The filters were washedunder low stringency (Example I.C.). Three positive plaques wereidentified one of which was IMR32 1.86. IMR32 1.86 was plaque purified,subcloned, and characterized by restriction mapping and DNA sequencing.IMR32 1.86 encodes α_(1C) sequences as described in Example II.B.1.Characterization by DNA sequencing revealed that IMR32 1.86 contains a73 nt deletion compared to the DNA encoding rabbit cardiac musclecalcium channel α₁ subunit [Mikami et al. (1989) Nature 340:230], nt2191 to 2263. These missing nucleotides correspond to nt 2176-2248 ofSEQ ID No. 3. Because the 5′-end of CNS 1.30 overlaps the 3′-end ofIMR32 1.86, some of these missing nucleotides, i.e., nt 2205-2248 of SEQID No. 3, are accounted for by CNS 1.30. The remaining missingnucleotides of the 73 nucleotide deletion in IMR32 1.86 (i.e., nt2176-2204 SEQ ID No. 3) were determined by PCR analysis ofdbcAMP-induced IMR32 cell RNA. The 73 nt deletion is a frame-shiftmutation and, thus, needs to be corrected. The exact human sequencethrough this region, (which has been determined by the DNA sequence ofCNS 1.30 and PCR analysis of IMR32 cell RNA) can be inserted into IMR321.86 by standard methods, e.g., replacement of a restriction fragment orsite-directed mutagenesis.

f. IMR32 1.157

One million recombinants of IMR32 cDNA library #4 (Example I.B.4.) werescreened with an XhoI-EcoRI fragment of IMR32 1.67 encoding α_(1C) nt 50to 774 (SEQ ID No. 3). The hybridization was performed using standardhybridization conditions (Example I. C.). The filters were washed underhigh stringency (Example I.C.). One of the positive plaques identifiedwas IMR32 1.157. This plaque was purified, the insert was restrictionmapped and subcloned to a standard plasmid vector pGEM7Z (Promega,Madison, Wis.). The DNA was characterized by sequencing. IMR32 1.157appears to encodes an alternative 5′ portion of the α₁ sequencebeginning with nt 1 to 89 (SEQ ID No. 5) and followed by nt 1 to 873(SEQ ID No. 3). Analysis of the 1.66 and 1.157 5′ sequence is describedbelow (Example II.B.3.).

3. Characterization of the α_(1C) Initiation of Translation Site

Portions of the sequences of IMR32 1.157 (nt 57 to 89, SEQ ID No. 5; nt1 to 67, SEQ ID No. 3), IMR32 1.66 (nt 100 to 132, SEQ ID No. 4; nt 1 to67, SEQ ID No. 3), were compared to the rabbit lung CaCB-receptor cDNAsequence, nt −33 to 67 [Biel et al. (1990) FEBS Lett. 269:409]. Thehuman sequences are possible alternative 5′ ends of the α_(1C)transcript encoding the region of initiation of translation. IMR32 1.66closely matches the CaCB receptor cDNA sequence and diverges from theCaCB receptor cDNA sequence in the 5′ direction beginning at nt 122 (SEQID No. 4). The start codon identified in the CaCB receptor cDNA sequenceis the same start codon used to describe the α_(1C) coding sequence, nt1 to 3 (SEQ ID No. 3). The functional significance of the IMR32 1.157sequence, nt 1 to 89 (SEQ ID No. 5), is not clear. Chimeras containingsequence between 1.157 and the α_(1C) coding sequence can be constructedand functional differences can be tested.

C. Isolation of Partial cDNA Clones Encoding the α_(1B) Subunit andConstruction of a Full-length Clone

A human basal ganglia cDNA library was screened with the rabbit skeletalmuscle α₁ subunit cDNA fragments (see Example II.A.2.a for descriptionof fragments) under low stringency conditions. One of the hybridizingclones was used to screen an IMR32 cell cDNA library to obtainadditional partial α_(1B) cDNA clones, which were in turn used tofurther screen an IMR32 cell cDNA library for additional partial cDNAclones. One of the partial IMR32 α_(1B) clones was used to screen ahuman hippocampus library to obtain a partial α_(1B) clone encoding the3′ end of the α_(1B) coding sequence. The sequence of some of theregions of the partial cDNA clones was compared to the sequence ofproducts of PCR analysis of IMR32 cell RNA to determine the accuracy ofthe cDNA sequences.

PCR analysis of IMR32 cell RNA and genomic DNA using oligonucleotideprimers corresponding to sequences located 5′ and 3′ of the STOP codonof the DNA encoding the α_(1B) subunit revealed an alternatively splicedα_(1B)-encoding mRNA in IMR32 cells. This second mRNA product is theresult of differential splicing of the α_(1B) subunit transcript toinclude another exon that is not present in the mRNA corresponding tothe other 3′ α_(1B) cDNA sequence that was initially isolated. Todistinguish these splice variants of the α_(1B) subunit, the subunitencoded by a DNA sequence corresponding to the form containing theadditional exon is referred to as α_(1B-1) (SEQ ID No. 7), whereas thesubunit encoded by a DNA sequence corresponding to the form lacking theadditional exon is referred to as α_(1B-2) (SEQ ID No. 8). The sequenceof α_(1B-1) diverges from that of α_(1B-2) beginning at nt 6633 (SEQ IDNo. 7). Following the sequence of the additional exon in α_(1B-1) (nt6633-6819; SEQ ID No. 7), the α_(1B-1) and α_(1B-2) sequences areidentical (i.e., nt 6820-7362 in SEQ ID No. 7 and nt 6633-7175 in SEQ IDNo. 8). SEQ ID No. 7 and No. 8 set forth 143 nt of 5′ untranslatedsequence (nt 1-143) as well as 202 nt of 3′ untranslated sequence (nt7161-7362, SEQ ID No. 7) of the DNA encoding α_(1B-1) and 321 nt of 3′untranslated sequence (nt 6855-7175, SEQ ID No. 8) of the DNA encodingα_(1B-2).

PCR analysis of the IS6 region of the α_(1B) transcript revealed whatappear to be additional splice variants based on multiple fragment sizesseen on an ethidium bromide-stained agarose gel containing the productsof the PCR reaction.

A full-length α_(1B-1) cDNA clone designated pcDNA-α_(1B-1) was preparedin an eight-step process as follows.

Step 1

The SacI restriction site of pGEM3 (Promega, Madison, Wis.) wasdestroyed by digestion at the SacI site, producing blunt ends bytreatment with T4 DNA polymerase, and religation. The new vector wasdesignated pGEMΔSac.

Step 2

Fragment 1 (HindIII/KpnI; nt 2337 to 4303 of SEQ ID No. 7) was ligatedinto HindIII/KpnI digested pGEM3ΔSac to produce pα1.177HK.

Step 3

Fragment 1 has a 2 nucleotide deletion (nt 3852 and 3853 of SEQ ID No.7). The deletion was repaired by inserting a PCR fragment (fragment 2)of IMR32 RNA into pα1.177HK. Thus, fragment 2 (NarI/KpnI; nt 3828 to4303 of SEQ ID No. 7) was inserted into NarI/KpnI digested pα1.177HKreplacing the NarI/KpnI portion of fragment 1 and producingpα1.177HK/PCR.

Step 4

Fragment 3 (KpnI/KpnI; nt 4303 to 5663 of SEQ ID No. 7) was ligated intoKpnI digested pα1.177HK/PCR to produce pα1B5′K.

Step 5

Fragment 4 (EcoRI/HindIII; EcoRI adaptor plus nt 1 to 2337 of SEQ ID No.7) and fragment 5 (HindIII/XhoI fragment of pα1B5′K; nt 2337 to 5446 ofSEQ ID No. 7) were ligated together into EcoRI/XhoI digested pcDNA1(Invitrogen, San Diego, Calif.) to produce pα1B5′.

Step 6

Fragment 6 (EcoRI/EcoRI; EcoRI adapters on both ends plus nt 5749 to7362 of SEQ ID No. 7) was ligated into EcoRI digested pBluescript II KS(Stratagene, La Jolla, Calif.) with the 5′ end of the fragment proximalto the KpnI site in the polylinker to produce pα1.230.

Step 7

Fragment 7 (KpnI/XhoI; nt 4303 to 5446 of SEQ ID No. 7), and fragment 8(XhoI/CspI; nt 5446 to 6259 of SEQ ID No. 7) were ligated into KpnI/CspIdigested pα1.230 (removes nt 5749 to 6259 of SEQ ID No. 7 that wasencoded in pα1.230 and maintains nt 6259 to 7362 of SEQ ID No. 7) toproduce pα1B3′.

Step 8

Fragment 9 (SphI/XhoI; nt 4993 to 5446 of SEQ ID No. 7) and fragment 10(XhoI/XbaI of pα1B3′; nt 5446 to 7319 of SEQ ID No. 7) were ligated intoSphI/XbaI digested pα1B5′ (removes nt 4993 to 5446 of SEQ ID No. 7 thatwere encoded in pα1B5′ and maintains nt 1 to 4850 of SEQ ID No. 7) toproduce pcDNAα_(1B-1).

The resulting construct, pcDNAα_(1B-1), contains, in pCDNA1, afull-length coding region encoding α_(1B-1) (nt 144-7362, SEQ ID No. 7),plus 5′ untranslated sequence (nt 1-143, SEQ ID No. 7) and 3′untranslated sequence (nt 7161-7319, SEQ ID No. 7) under thetranscriptional control of the CMV promoter.

D. Isolation of DNA Encoding Human Calcium Channel α_(1A) Subunits

1. Isolation of Partial Clones

DNA clones encoding portions of human calcium channel α_(1A) subunitswere obtained by hybridization screening of human cerebellum cDNAlibraries and nucleic acid amplification of human cerebellum RNA. Clonescorresponding to the 3′ end of the α_(1A) coding sequence were isolatedby screening 1×10⁶ recombinants of a randomly primed cerebellum cDNAlibrary (size-selected for inserts greater than 2.8 kb in length) underlow stringency conditions (6×SSPE, 5×Denhart's solution, 0.2% SDS, 200μg/ml sonicated herring sperm DNA, 42° C.) with oligonucleotide 704containing nt 6190-6217 of the rat α_(1A) coding sequence [Starr et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 88:5621-5625]. Washes wereperformed under low stringency conditions. Several clones thathybridized to the probe (clones 60 1.251-α1.259 and α1.244) werepurified and characterized by restriction enzyme mapping and DNAsequence analysis. At least two of the clones, α1.244 and α1.254,contained a translation termination codon. Although clones α1.244 andα1.254 are different lengths, they both contain a sequence ofnucleotides that corresponds to the extreme 3′ end of the α_(1A)transcript, i.e., the two clones overlap. These two clones are identicalin the region of overlap, except, clone α1.244 contains a 5 and 12nucleotides that are not present in α1.254.

To obtain additional α_(1A)-encoding clones, 1×10⁶ recombinants of arandomly primed cerebellum cDNA library (size-selected for insertsranging from 1.0 to 2.8 kb in length) was screened for hybridization tothree oligonucleotides: oligonucleotide 701 (containing nucleotides2288-2315 of the rat α_(1A) coding sequence), oligonucleotide 702(containing nucleotides 3559-3585 of the rat α_(1A) coding sequence) andoligonucleotide 703 (containing nucleotides 4798-4827 of the rat α_(1A)coding sequence). Hybridization and washes were performed using the sameconditions as used for the first screening with oligonucleotide 704,except that washes were conducted at 45° C. Twenty clones (clonesα1.269-α1.288) hybridized to the probe. Several clones wereplaque-purified and characterized by restriction enzyme mapping and DNAsequence analysis. One clone, α1.279, contained about 170 nucleotidesthat is not present in other clones corresponding to the same region ofthe coding sequence. This region may be present in other splice variant.None of the clones contained a translation intiation codon.

To obtain clones corresponding to the 5′ end of the human α_(1A) codingsequence, another cerebellum cDNA library was prepared usingoligonucleotide 720 (containing nucleotides 2485-2510 of SEQ ID No. 22to specifically prime first-strand cDNA synthesis. The library (8×10⁵recombinants) was screened for hybridization to three oligonucleotides:oligonucleotide 701, oligonucleotide 726 (containing nucleotides2333-2360 of the rat α_(1A) coding sequence) and oligonucleotide 700(containing nucleotides 767-796 of the rat α_(1A) coding sequence) underlow stringency hybridization and washing conditions. Approximately 50plaques hybridized to the probe. Hybridizing clones α1.381-α1.390 wereplaque-purified and characterized by restriction enzyme maping and DNAsequence analysis. At least one of the clones, α1.381, contained atranslation initiation codon.

Alignment of the sequences of the purified clones revealed that thesequences overlapped to comprise the entire α_(1A) coding sequence.However, not all the overlapping sequences of partial clones containedconvenient enzyme restriction sites for use in ligating partial clonesto construct a full-length α_(1A) coding sequence. To obtain DNAfragments containing convenient restriction enzyme sites that could beused in constructing a full-length α_(1A) DNA, cDNA was synthesized fromRNA isolated from human cerebellum tissue and subjected to nucleic acidamplification. The oligonucleotides used as primers corresponded tohuman α_(1A) coding sequence located 5′ and 3′ of selected restrictionenzyme sites. Thus, in the first amplification reaction,oligonucleotides 753 (containing nucleotides 2368-2391 of SEQ ID No. 22)and 728 (containing nucleotides 3179-3202 of SEQ ID No. 22) were used asthe primer pair. To provide a sufficient amount of the desired DNAfragment, the product of this amplification was reamplified usingoligonucleotides 753 and 754 (containing nucleotides 3112-3135 of SEQ IDNo. 22 as the primer pair. The resulting product was 768 bp in length.In the second amplification reaction, oligonucleotides 719 (containingnucleotides 4950-4975 of SEQ ID No. 22 and 752 (containing nucleotides5647-5670 of SEQ ID No. 22) were used as the primer pair. To provide asufficient amount of the desired second DNA fragment, the product ofthis amplification was reamplified using oligonucleotides 756(containing nucleotides 5112-5135 of SEQ ID No. 22) and 752 as theprimer pair. The resulting product was 559 bp in length.

2. Construction of Full-Length α_(1A) Coding Sequences

Portions of clone α1.381, the 768-bp nucleic acid amplification product,clone α1.278, the 559-bp nucleic acid amplification product, and cloneα1.244 were ligated at convenient restriction sites to generate afull-length α_(1A) coding sequence referred to as α_(1A-1).

Comparison of the results of sequence analysis of clones α1.244 andα1.254 indicated that the primary transcript of the α_(1A) subunit geneis alternatively spliced to yield at least two variant mRNAs encodingdifferent forms of the α_(1A) subunit. One form, α_(1A-1), is encoded bythe sequence shown in SEQ ID No. 22. The sequence encoding a secondform, α_(1A-2), differs from the α_(1A-1)-encoding sequence at the 3′end in that it lacks a 5-nt sequence found in clone α1.244 (nucleotides7035-7039 of SEQ ID No. 22). This deletion shifts the reading frame andintroduces a translation termination codon resulting in an α_(1A-2)coding sequence that encodes a shorter α_(1A) subunit than that encodedby the α_(1A-1) splice variant. Consequently, a portion of the 3′ end ofthe α_(1A-1) coding sequence is actually 3′ untranslated sequence in theα_(1A-2) DNA. The complete sequence of α_(1A-12), which can beconstructed by ligating portions of clone α1.381, the 768-bp nucleicacid amplification product, clone α1.278, the 559-bp nucleic acidamplification product and clone α1.254, is set forth in SEQ ID No. 23.

E. Isolation of DNA Encoding the α_(1E) Subunit

DNA encoding α_(1E) subunits of the human calcium channel were isolatedfrom human hippocampus libraries. The selected clones sequenced. DNAsequence analysis of DNA clones encoding the α_(1B) subunit indicatedthat at least two alternatively spliced forms of the same α_(1E) subunitprimary transcript are expressed. One form has the sequence set forth inSEQ ID No. 24 and was designated α_(1E-1) and the other was designatedα_(1E-3), which has the sequence obtained by inserting SEQ ID No. 25between nucleotides 2405 and 2406 of SEQ ID No. 24. The resultingsequence of α_(1E-3) is set forth in SEQ ID No. 27.

The subunit designated α_(1E-1) has a calculated molecular weight of254,836 and the subunit designated α_(1E-3) has a calculated molecularweight of 257,348. α_(1E-3) has a 19 amino acid insertion (encoded bySEQ ID No. 25) relative to α_(1E-1) in the region that appears to be thecytoplasmic loop between transmembrane domains IIS6 and IIIS1.

Example III

Isolation of cDNA Clones Encoding the Human Neuronal Calcium Channel β₁Subunit

A. Isolation of Partial cDNA Clones Encoding the β Subunit andConstruction of a Full-length Clone Encoding the β₁ Subunit

A human hippocampus cDNA library was screened with the rabbit skeletalmuscle calcium channel β₁ subunit cDNA fragment (nt 441 to 1379) [forisolation and sequence of the rabbit skeletal muscle calcium channel β₁subunit cDNA, see U.S. patent application Ser. No. 482,384 or Ruth etal. (1989) Science 245:1115] using standard hybridization conditions(Example I.C.). A portion of one of the hybridizing clones was used torescreen the hippocampus library to obtain additional cDNA clones. ThecDNA inserts of hybridizing clones were characterized by restrictionmapping and DNA sequencing and compared to the rabbit skeletal musclecalcium channel β₁ subunit cDNA sequence.

Portions of the partial β₁ subunit cDNA clones were ligated to generatea full-length clone encoding the entire β₁ subunit. SEQ ID No. 9 showsthe β₁ subunit coding sequence (nt 1-1434) as well as a portion of the3′ untranslated sequence (nt 1435-1546). The deduced amino acid sequenceis also provided in SEQ ID No. 9. In order to perform expressionexperiments, full-length β₁ subunit cDNA clones were constructed asfollows.

Step 1

DNA fragment 1 (˜800 bp of 5′ untranslated sequence plus nt 1-277 of SEQID No. 9) was ligated to DNA fragment 2 (nt 277-1546 of SEQ ID No. 9plus 448 bp of intron sequence) and cloned into pGEM7Z. The resultingplasmid, pβ1-1.18, contained a full-length β₁ subunit clone thatincluded a 448-bp intron.

Step 2

To replace the 5′ untranslated sequence of pβ1-1.18 with a ribosomebinding site, a double-stranded adapter was synthesized that contains anEcoRI site, sequence encoding a ribosome binding site (5′-ACCACC-3′) andnt 1-25 of SEQ ID No. 9. The adapter was ligated to SmaI-digestedpβ1-1.18, and the products of the ligation reaction were digested withEcoRI.

Step 3

The EcoRI fragment from step 2 containing the EcoRI adapter, efficientribosome binding site and nt 1-1546 of SEQ ID No. 9 plus intron sequencewas cloned into a plasmid vector and designated pβ1-1.18RBS. The EcoRIfragment of pβ1-1.18RBS was subcloned into EcoRI-digested pcDNA1 withthe initiation codon proximal to CMV promoter to formpHBCaCHβ_(1a)RBS(A).

Step 4

To generate a full-length clone encoding the β₁ subunit lacking intronsequence, DNA fragment 3 (nt 69-1146 of SEQ ID No. 9 plus 448 bp ofintron sequence followed by nt 1147-1546 of SEQ ID No. 9), was subjectedto site-directed mutagenesis to delete the intron sequence, therebyyielding pβ1(−). The EcoRI-XhoI fragment of pβ1-1.18RBS (containing ofthe ribosome binding site and nt 1-277 of SEQ ID No. 9) was ligated tothe XhoI-EcoRI fragment of pβ1(−) (containing of nt 277-1546 of SEQ IDNo. 9) and cloned into pcDNA1 with the initiation of translationproximal to the CMV promoter. The resulting expression plasmid wasdesignated pHBCaCHβ_(1b)RBS(A).

B. Splice Variant β₁₋₃

DNA sequence analysis of the DNA clones encoding the β₁ subunitindicated that in the CNS at least two alternatively spliced forms ofthe same human β₁ subunit primary transcript are expressed. One form isrepresented by the sequence shown in SEQ ID No. 9 and is referred to asβ₁₋₂. The sequences of β₁₋₂ and the alternative form, β₁₋₃, diverge atnt 1334 (SEQ ID No. 9). The complete β₁₋₃ sequence (nt 1-1851),including 3′ untranslated sequence (nt 1795-1851), is set forth in SEQID No. 10.

Example IV

Isolation of cDNA Clones Encoding the Human Neuronal Calcium Channelα₂-subunit

A. Isolation of cDNA Clones

The complete human neuronal α₂ coding sequence (nt 35-3307) plus aportion of the 5′ untranslated sequence (nt 1 to 34) as well as aportion of the 3′ untranslated sequence (nt 3308-3600) is set forth inSEQ ID No. 11.

To isolate DNA encoding the human neuronal α₂ subunit, human α₂ genomicclones first were isolated by probing human genomic Southern blots usinga rabbit skeletal muscle calcium channel α₂ subunit cDNA fragment [nt 43to 272, Ellis et al. (1988) Science 240:1661]. Human genomic DNA wasdigested with EcoRI, electrophoresed, blotted, and probed with therabbit skeletal muscle probe using standard hybridization conditions(Example I.C.) and low stringency washing conditions (Example I.C.). Tworestriction fragments were identified, 3.5 kb and 3.0 kb. These EcoRIrestriction fragments were cloned by preparing a λgt11 librarycontaining human genomic EcoRI fragments ranging from 2.2 kb to 4.3 kb.The library was screened as described above using the rabbit α₂ probe,hybridizing clones were isolated and characterized by DNA sequencing.HGCaCHα2.20 contained the 3.5 kb fragment and HGCaCHα2.9 contained the3.0 kb fragment.

Restriction mapping and DNA sequencing revealed that HGCaCHα2.20contains an 82 bp exon (nt 130 to 211 of the human α₂ coding sequence,SEQ ID No. 11) on a 650 bp PstI-XbaI restriction fragment and thatHGCaCHα2.9 contains 105 bp of an exon (nt 212 to 316 of the codingsequence, SEQ ID No. 11) on a 750 bp XbaI-BGlII restriction fragment.These restriction fragments were used to screen the human basal gangliacDNA library (Example II.C.2.a.). HBCaCHα2.1 was isolated (nt 29 to1163, SEQ ID No. 11) and used to screen a human brain stem cDNA library(ATCC Accession No. 37432) obtained from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852. Two clones wereisolated, HBCaCHα2.5 (nt 1 to 1162, SEQ ID No. 11) and HBCaCHα2.8 (nt714 to 1562, SEQ ID No. 11, followed by 1600 nt of interveningsequence). A 2400 bp fragment of HBCaCHα2.8 (beginning at nt 759 of SEQID No. 11 and ending at a SmaI site in the intron) was used to rescreenthe brain stem library and to isolate HBCaCHα2.11 (nt 879 to 3600, SEQID No. 11). Clones HBCaCHα2.5 and HBCaCHα2.11 overlap to encode anentire human brain α₂ protein.

B. Construction of pHBCaCHα₂A

To construct pHBCaCHα₂A containing DNA encoding a full-length humancalcium channel α₂ subunit, an (EcoRI)-PvuII fragment of HBCaCHα2.5 (nt1 to 1061, SEQ ID No. 11, EcoRI adapter, PvuII partial digest) and aPvuII-PstI fragment of HBCaCHα2.11 (nt 1061 to 2424 SEQ ID No. 11; PvuIIpartial digest) were ligated into EcoRI-PstI-digested pIBI24(Stratagene, La Jolla, Calif.). Subsequently, an (EcoRI)-PstI fragment(nt 1 to 2424 SEQ ID No. 11) was isolated and ligated to a PstI-(EcoRI)fragment (nt 2424 to 3600 SEQ ID No. 11) of HBCaCHα2.11 inEcoRI-digested pIBI24 to produce DNA, HBCaCHα2, encoding a full-lengthhuman brain α₂ subunit. The 3600 bp EcoRI insert of HBCaCHα2 (nt 1 to3600, SEQ ID No. 11) was subcloned into pcDNA1 (pHBCaCHα2A) with themethionine initiating codon proximal to the CMV promoter. The 3600 bpEcoRI insert of HBCaCHα2 was also subcloned into pSV2dHFR [Subramani etal. (1981). Mol. Cell. Biol. 1:854-864] which contains the SV40 earlypromoter, mouse dihydrofolate reductase (dhfr) gene, SV40polyadenylation and splice sites and sequences required for maintenanceof the vector in bacteria.

Example V

Differential Processing of the Human β₁ Transcript and the Human α₂Transcript

A. Differential Processing of the β₁ Transcript

PCR analysis of the human β₁ transcript present in skeletal muscle,aorta, hippocampus and basal ganglia, and HEK 293 cells revealeddifferential processing of the region corresponding to nt 615-781 of SEQID No. 9 in each of the tissues. Four different sequences that result infive different processed transcripts through this region wereidentified. The β₁ transcripts from the different tissues containeddifferent combinations of the four sequences, except for one of the β₁transcripts expressed in HEK 293 cells (β₁₋₅) which lacked all foursequences.

None of the β₁ transcripts contained each of the four sequences;however, for ease of reference, all four sequences are set forthend-to-end as a single long sequence in SEQ ID No. 12. The foursequences that are differentially processed are sequence 1 (nt 14-34 inSEQ ID No. 12), sequence 2 (nt 35-55 in SEQ ID No. 12), sequence 3 (nt56-190 in SEQ ID No. 12) and sequence 4 (nt 191-271 in SEQ ID No. 12).The forms of the β₁ transcript that have been identified include: (1) aform that lacks sequence 1 called β₁₋₁ (expressed in skeletal muscle),(2) a form that lacks sequences 2 and 3 called β₁₋₂ (expressed in CNS),(3) a form that lacks sequences 1, 2 and 3 called β₁₋₄ (expressed inaorta and HEK cells) and (4) a form that lacks sequences 1-4 called β₁₋₅(expressed in HEK cells). Additionally, the β₁₋₄ and β₁₋₅ forms containthe guanine nucleotide (nt 13 in SEQ ID No. 12) which is absent in theβ₁₋₁ and β₁₋₂ forms. The sequences of these splice variants designatedβ₁₋₁, β₁₋₂, β₁₋₃, β₁₋₄ and β₁₋₅ are set forth in Sequence ID Nos. 32, 9,10, 33 and 34, respectively.

B. Differential Processing of Transcripts Encoding the α₂ Subunit

The complete human neuronal α₂ coding sequence (nt 35-3307) plus aportion of the 5′ untranslated sequence (nt 1 to 34) as well as aportion of the 3′ untranslated sequence (nt 3308-3600) is set forth asSEQ ID No. 11.

PCR analysis of the human α₂ transcript present in skeletal muscle,aorta, and CNS revealed differential processing of the regioncorresponding to nt 1595-1942 of SEQ ID No. 11 in each of the tissues.

The analysis indicated that the primary transcript of the genomic DNAthat includes the nucleotides corresponding to nt 1595-1942 alsoincludes an additional sequence (SEQ ID No. 13:5′CCTATTGGTGTAGGTATACCAACAATTAATTTAAGAAAAAGGAGACCCAATATCCAG 3′) insertedbetween nt 1624 and 1625 of SEQ ID No. 11. Five alternatively splicedvariant transcripts that differ in the presence or absence of one tothree different portions of the region of the primary transcript thatincludes the region of nt 1595-1942 of SEQ ID No. 11 plus SEQ ID No. 13inserted between nt 1624 and 1625 have been identified. The fiveα₂-encoding transcripts from the different tissues include differentcombinations of the three sequences, except for one of the α₂transcripts expressed in aorta which lacks all three sequences. None ofthe α₂ transcripts contained each of the three sequences. The sequencesof the three regions that are differentially processed are sequence 1(SEQ ID No. 13), sequence 2 (5′ AACCCCAAATCTCAG 3′, which is nt1625-1639 of SEQ ID No. 11), and sequence 3 (5′ CAAAAAAGGGCAAAATGAAGG3′, which is nt 1908-1928 of SEQ ID No. 11). The five α₂ formsidentified are (1) a form that lacks sequence 3 called α_(2a) (expressedin skeletal muscle), (2) a form that lacks sequence 1 called α_(2b)(expressed in CNS), (3) a form that lacks sequences 1 and 2 calledα_(2c) expressed in aorta), (4) a form that lacks sequences 1, 2 and 3called α_(2d) (expressed in aorta) and (5) a form that lacks sequences 1and 3 called α_(2e) (expressed in aorta). The sequences of each of thesesubunits are set forth in sequence ID NOs. 11 (α_(2b)), SEQ ID NO. 28(α_(2a)) SEQ ID NO. 29 (α_(2c)), SEQ ID NO. 30 (α_(2d)), and SEQ ID NO.31 (α_(2e)).

Example VI

Isolation of DNA Encoding a Calcium Channel γ Subunit from a Human BraincDNA Library

A. Isolation of DNA Encoding the γ Subunit

Approximately 1×10⁶ recombinants from a λgtll-based human hippocampuscDNA library (Clontech catalog #HL1088b, Palo Alto, Calif.) werescreened by hybridization to a 484 bp sequence of the rabbit skeletalmuscle calcium channel γ subunit cDNA (nucleotides 621-626 of the codingsequence plus 438 nucleotides of 3′-untranslated sequence) contained invector γJ10 [Jay, S. et al. (1990). Science 248:490-492]. Hybridizationwas performed using moderate stringency conditions (20% deionizedformamide, 5×Denhardt's, 6×SSPE, 0.2% SDS, 20 μg/ml herring sperm DNA,42° C.) and the filters were washed under low stringency (see ExampleI.C.). A plaque that hybridized to this probe was purified and insertDNA was subcloned into pGEM7Z. This cDNA insert was designated γ1.4.

B. Characterization of γ1.4

γ1.4 was confirmed by DNA hybridization and characterized by DNAsequencing. The 1500 bp SstI fragment of γ1.4 hybridized to the rabbitskeletal muscle calcium channel γ subunit cDNA γJ10 on a Southern blot.SEQ analysis of this fragment revealed that it contains of approximately500 nt of human DNA sequence and ˜1000 nt of λgtll sequence (includeddue to apparent destruction of one of the EcoRI cloning sites in λgt11).The human DNA sequence contains of 129 nt of coding sequence followedimmediately by a translational STOP codon and 3′ untranslated sequence(SEQ ID No. 14).

To isolate the remaining 5′ sequence of the human γ subunit cDNA, humanCNS cDNA libraries and/or preparations of mRNA from human CNS tissuescan first be assayed by PCR methods using oligonucleotide primers basedon the γ cDNA-specific sequence of γ14. Additional human neuronal γsubunit-encoding DNA can isolated from cDNA libraries that, based on theresults of the PCR assay, contain γ-specific amplifiable cDNA.Alternatively, cDNA libraries can be constructed from mRNA preparationsthat, based on the results of PCR assays, contain γ-specific amplifiabletranscripts. Such libraries are constructed by standard methods usingoligo dT to prime first-strand cDNA synthesis from poly A⁺ RNA (seeExample I.B.). Alternatively, first-strand cDNA can be specified bypriming first-strand cDNA synthesis with a δ cDNA-specificoligonucleotide based on the human DNA sequence in γ1.4. A cDNA librarycan then be constructed based on this first-strand synthesis andscreened with the γ-specific portion of γ1.4.

Example VII

Isolation of cDNA Clones Encoding the Human Neuronal Ca Channel β₂Subunit

Isolation of DNA Encoding Human Calcium Channel β₂ Subunits

Sequencing of clones isolated as described in Example III revealed aclone encoding a substantial portion of a human neuronal calcium channelβ₂ subunit (designated β_(2D) see, nucleotides 1-1866 SEQ ID No. 26). Anoligonucleotide based on the 5′ end of this clone was used to prime ahuman hippocampus cDNA library. The library was screened with this β₂clone under conditions of low to medium stringency (final wash 0.5×SSPE,50° C.). Several hybridizing clones were isolated and sequenced. Amongthese clones were those that appear to encode β_(2C),β_(2E) and β_(2F).

A randomly primed hoppocampus library was then screened using acombination of the clone encoding β_(2D) and a portion of the β₃ clonedeposited under ATCC Accession No. 69048. Multiple hybridizing cloneswere isolated. Among were clones designated β101, β102 and β104. β101appears to encodes the 5′ end of a splice variant of β₂ , designated_(2E). β102 and β104 encode portions of the 3′ end of β₂.

It appears that the β₂ splice variants include nucleotide 182-2264 ofSEQ ID No. 26 and differ only between the start codon and nucleotidesthat correspond to 182 of SEQ. ID No. 26.

EXAMPLE VIII

Isolation of cDNA Clones Encoding the Human Neuronal Ca Channel β₃Subunit

Sequencing of clones isolated as described in Example III also revealeda clone encoding a human neuronal calcium channel β₃ subunit. This cloneincludes nucleotides having the sequence set forth in SEQ ID Nos. 19 and20 and also includes DNA that has been deposited as plasmid β1.42 (ATCCAccession No. 69048).

EXAMPLE IX

Recombinant Expression of Human Neuronal Calcium Channel Subunit-ncodingcDNA and RNA Transcripts in Mammalian Cells

A. Recombinant Expression of the Human Neuronal Calcium Channel α₂Subunit cDNA in DG44 Cells

1. Stable Transfection of DG44 Cells

DG44 cells [dhfr⁻ Chinese hamster ovary cells; see, e.g., Urlaub, G. etal. (1986) Som. Cell Molec. Genet. 12:555-566] obtained from LawrenceChasin at Columbia University were stably transfected by CaPO₄precipitation methods [Wigler et al. (1979) Proc. Natl. Acad. Sci. USA76:1373-1376) with pSV2dhfr vector containing the human neuronal calciumchannel α₂-subunit cDNA (see Example IV) for polycistronicexpression/selection in transfected cells. Transfectants were grown on10% DMEM medium without hypoxanthine or thymidine in order to selectcells that had incorporated the expression vector. Twelve transfectantcell lines were established as indicated by their ability to survive onthis medium.

2. Analysis of α₂ Subunit cDNA Expression in Transfected DG44 Cells

Total RNA was extracted according to the method of Birnboim [(1988) Nuc.Acids Res. 16:1487-1497] from four of the DG44 cell lines that had beenstably transfected with pSV2dhfr containing the human neuronal calciumchannel α₂ subunit cDNA. RNA (˜15 μg per lane) was separated on a 1%agarose formaldehyde gel, transferred to nitrocellulose and hybridizedto the random-primed human neuronal calcium channel α₂ cDNA(hybridization: 50% formamide, 5×SSPE, 5×Denhardt's, 42° C.; wash:0.2×SSPE, 0.1% SDS, 65° C.). Northern blot analysis of total RNA fromfour of the DG44 cell lines that had been stably transfected withpSV2dhfr containing the human neuronal calcium channel α₂ subunit cDNArevealed that one of the four cell lines contained hybridizing mRNA thesize expected for the transcript of the α₂ subunit cDNA (5000 nt basedon the size of the cDNA) when grown in the presence of 10 mM sodiumbutyrate for two days. Butyrate nonspecifically induces transcriptionand is often used for inducing the SV40 early promoter (Gorman, C. andHoward, B. (1983) Nucleic Acids Res. 11:1631]. This cell line, 44α₂-9,also produced mRNA species smaller (several species) and larger (6800nt) than the size expected for the transcript of the α₂ cDNA (5000 nt)that hybridized to the α₂ cDNA-based probe. The 5000- and 6800-nttranscripts produced by this transfectant should contain the entire α₂subunit coding sequence and therefore should yield a full-length α₂subunit protein. A weakly hybridizing 8000-nucleotide transcript waspresent in untransfected and transfected DG44 cells. Apparently, DG44cells transcribe a calcium channel α₂ subunit or similar gene at lowlevels. The level of expression of this endogenous α₂ subunit transcriptdid not appear to be affected by exposing the cells to butyrate beforeisolation of RNA for northern analysis.

Total protein was extracted from three of the DG44 cell lines that hadbeen stably transfected with pSV2dhfr containing the human neuronalcalcium channel α₂ subunit cDNA. Approximately 10⁷ cells were sonicatedin 300 μl of a solution containing 50 mM HEPES, 1 mM EDTA, 1 mM PMSF. Anequal volume of 2×loading dye [Laemmli, U. K. (1970). Nature 227:680]was added to the samples and the protein was subjected toelectrophoresis on an 8% polyacrylamide gel and then electrotransferredto nitrocellulose. The nitrocellulose was incubated with polyclonalguinea pig antisera (1:200 dilution) directed against the rabbitskeletal muscle calcium channel α₂ subunit (obtained from K. Campbell,University of Iowa) followed by incubation with [¹²⁵I]-protein A. Theblot was exposed to X-ray film at −70° C. Reduced samples of proteinfrom the transfected cells as well as from untransfected DG44 cellscontained immunoreactive protein of the size expected for the α₂ subunitof the human neuronal calcium channel (130-150 kDa). The level of thisimmunoreactive protein was higher in 44α₂-9 cells that had been grown inthe presence of 10 mM sodium butyrate than in 44α₂-9 cells that weregrown in the absence of sodium butyrate. These data correlate well withthose obtained in northern analyses of total RNA from 44α₂-9 anduntransfected DG44 cells. Cell line 44α₂-9 also produced a 110 kDimmunoreactive protein that may be either a product of proteolyticdegradation of the full-length α₂ subunit or a product of translation ofone Qf the shorter (<5000 nt) mRNAs produced in this cell line thathybridized to the α₂ subunit cDNA probe.

B. Expression of DNA Encoding Human Neuronal Calcium Channel α₁, α₂ andβ₁ Subunits in HEK Cells

Human embryonic kidney cells (HEK 293 cells) were transiently and stablytransfected with human neuronal DNA encoding calcium channel subunits.Individual transfectants were analyzed electrophysiologically for thepresence of voltage-activated barium currents and functional recombinantvoltage-dependent calcium channels were.

1. Transfection of HEK 293 Cells

Separate expression vectors containing DNA encoding human neuronalcalcium channel α_(1D), α₂ and β₁ subunits, plasmids pVDCCIII(A),pHBCaCHα₂A, and pHBCaCHβ_(1a)RBS(A), respectively, were constructed asdescribed in Examples II.A.3, IV.B. and III.B.3., respectively. Thesethree vectors were used to transiently co-transfect HEK 293 cells. Forstable transfection of HEK 293 cells, vector pHBCaCHβ_(1b)RBS(A)(Example III.B.3.) was used in place of pHBCaCHβ_(1a)RBS(A) to introducethe DNA encoding the β₁ subunit into the cells along with pVDCCIII(A)and pHBCaCHα₂A.

a. Transient Transfection

Expression vectors pVDCCIII(A), pHBCaCHα₂A and pHBCaCHβ_(1a)RBS(A) wereused in two sets of transient transfections of HEK 293 cells (ATCCAccession No. CRL1573). In one transfection procedure, HEK 293 cellswere transiently cotransfected with the α₁ subunit cDNA expressionplasmid, the α₂ subunit cDNA expression plasmid, the β₁ subunit cDNAexpression plasmid and plasmid pCMVβgal (Clontech Laboratories, PaloAlto, Calif.). Plasmid pCMVβgal contains the lacZ gene (encoding E. coliβ-galactosidase) fused to the cytomegalovirus (CMV) promoter and wasincluded in this transfection as a marker gene for monitoring theefficiency of transfection. In the other transfection procedure, HEK 293cells were transiently co-transfected with the α₁ subunit cDNAexpression plasmid pVDCCIII(A) and pCMVβgal. In both transfections,2-4×10⁶ HEK 293 cells in a 10-cm tissue culture plate were transientlyco-transfected with 5 μg of each of the plasmids included in theexperiment according to standard CaPO₄ precipitation transfectionprocedures (Wigler et al. (1979) Proc. Natl. Acad. Sci. USA76:1373-1376). The transfectants were analyzed for β-galactosidaseexpression by direct staining of the product of a reaction involvingβ-galactosidase and the X-gal substrate [Jones, J. R. (1986) EMBO5:3133-3142] and by measurement of β-galactosidase activity [Miller, J.H. (1972) Experiments in Molecular Genetics, pp. 352-355, Cold SpringHarbor Press]. To evaluate subunit cDNA expression in thesetransfectants, the cells were analyzed for subunit transcript production(northern analysis), subunit protein production (immunoblot analysis ofcell lysates) and functional calcium channel expression(electrophysiological analysis).

b. Stable Transfection

HEK 293 cells were transfected using the calcium phosphate transfectionprocedure [Current Protocols in Molecular Biology, Vol. 1, WileyInter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)]. Ten-cm plates,each containing one-to-two million HEK 293 cells, were transfected with1 ml of DNA/calcium phosphate precipitate containing 5 μg pVDCCIII(A), 5μg pHBCaCHα₂A, 5 μg pHBCaCHβ_(1bRBS(A),) 5 μg pCMVBgal and 1 μpSV2neo(as a selectable marker). After 10-20 days of growth in media containing500 μg G418, colonies had formed and were isolated using cloningcylinders.

2. Analysis of HEK 293 Cells Transiently Transfected with DNA EncodingHuman Neuronal Calcium Channel Subunits

a. Analysis of β-galactosidase Expression

Transient transfectants were assayed for β-galactosidase expression byβ-galactosidase activity assays (Miller, J. H., (1972) Experiments inMolecular Genetics, pp. 352-355, Cold Spring Harbor Press) of celllysates (prepared as described in Example VII.A.2) and staining of fixedcells (Jones, J. R. (1986) EMBO 5:3133-3142). The results of theseassays indicated that approximately 30% of the HEK 293 cells had beentransfected.

b. Northern Analysis

PolyA+ RNA was isolated using the Invitrogen Fast Trak Kit (InVitrogen,San Diego, Calif.) from HEK 293 cells transiently transfected with DNAencoding each of the α₁, α₂ and β₁ subunits and the lacZ gene or the α₁subunit and the lacZ gene. The RNA was subjected to electrophoresis onan agarose gel and transferred to nitrocellulose. The nitrocellulose wasthen hybridized with one or more of the following radiolabeled probes:the lacZ gene, human neuronal calcium channel α_(1D) subunit-encodingcDNA, human neuronal calcium channel α₂ subunit-encoding cDNA or humanneuronal calcium channel β₁ subunit-encoding cDNA. Two transcripts thathybridized with the α₁ subunit-encoding cDNA were detected in HEK 293cells transfected with the DNA encoding the α₁, α₂, and β₁ subunits andthe lacZ gene as well as in HEK 293 cells transfected with the α₁subunit cDNA and the lacZ gene. One mRNA species was the size expectedfor the transcript of the α₁ subunit cDNA (8000 nucleotides). The secondRNA species was smaller (4000 nucleotides) than the size expected forthis transcript. RNA of the size expected for the transcript of the lacZgene was detected in cells transfected with the α₁, α₂ and β₁subunit-encoding cDNA and the lacZ gene and in cells transfected withthe α₁ subunit cDNA and the lacZ gene by hybridization to the lacZ genesequence.

RNA from cells transfected with the α₁, α₂ and β₁ subunit-encoding cDNAand the lacz gene was also hybridized with the α₂ and β₁ subunit cDNAprobes. Two mRNA species hybridized to the α₂ subunit cDNA probe. Onespecies was the size expected for the transcript of the α₂ subunit cDNA(4000 nucleotides). The other species was larger (6000 nucleotides) thanthe expected size of this transcript. Multiple RNA species in the cellsco-transfected with α₁, α₂ and β₁ subunit-encoding cDNA and the lacZgene hybridized to the β₁ subunit cDNA probe. Multiple β-subunittranscripts of varying sizes were produced since the β subunit cDNAexpression vector contains two potential polyA⁺ addition sites.

c. Electrophysiological Analysis

Individual transiently transfected HEK 293 cells were assayed for thepresence of voltage-dependent barium currents using the whole-cellvariant of the patch clamp technique [Hamill et al. (1981). PflugersArch. 391:85-100]. HEK 293 cells transiently transfected with pCMVβgalonly were assayed for barium currents as a negative control in theseexperiments. The cells were placed in a bathing solution that containedbarium ions to serve as the current carrier. Choline chloride, insteadof NaCl or KCl, was used as the major salt component of the bathsolution to eliminate currents through sodium and potassium channels.The bathing solution contained 1 mM MgCl₂ and was buffered at pH 7.3with 10 mM HEPES (pH adjusted with sodium or tetraethylammoniumhydroxide). Patch pipettes were filled with a solution containing 135 mMCsCl, 1 mM MgCl₂, 10 mM glucose, 10 mM EGTA, 4 mM ATP and 10 mM HEPES(pH adjusted to 7.3 with tetraethylammonium hydroxide). Cesium andtetraethylammonium ions block most types of potassium channels. Pipetteswere coated with Sylgard (Dow-Corning, Midland, Mich.) and hadresistances of 1-4 megohm. Currents were measured through a 500 megohmheadstage resistor with the Axopatch IC (Axon Instruments, Foster City,Calif.) amplifier, interfaced with a Labmaster (Scientific Solutions,Solon, Ohio) data acquisition board in an IBM-compatible PC. PClamp(Axon Instruments) was used to generate voltage commands and acquiredata. Data were analyzed with pClamp or Quattro Professional (BorlandInternational, Scotts Valley, Calif.) programs.

To apply drugs, “puffer” pipettes positioned within several micrometersof the cell under study were used to apply solutions by pressureapplication. The drugs used for pharmacological characterization weredissolved in a solution identical to the bathing solution. Samples of a10 mM stock solution of Bay K 8644 (RBI, Natick, Mass.), which wasprepared in DMSO, were diluted to a final concentration of 1 μM in 15 mMBa²⁺-containing bath solution before they were applied.

Twenty-one negative control HEK 293 cells (transiently transfected withthe lacZ gene expression vector pCMVβgal only) were analyzed by thewhole-cell variant of the patch clamp method for recording currents.Only one cell displayed a discernable inward barium current; thiscurrent was not affected by the presence of 1 μM Bay K 8644. Inaddition, application of Bay K 8644 to four cells that did not displayBa²⁺ currents did not result in the appearance of any currents.

Two days after transient transfection of HEK 293 cells with α₁, α₂ andβ₁ subunit-encoding cDNA and the lacZ gene, individual transfectantswere assayed for voltage-dependent barium currents. The currents in ninetransfectants were recorded. Because the efficiency of transfection ofone cell can vary from the efficiency of transfection of another cell,the degree of expression of heterologous proteins in individualtransfectants varies and some cells do not incorporate or express theforeign DNA. Inward barium currents were detected in two of these ninetransfectants. In these assays, the holding potential of the membranewas −90 mV. The membrane was depolarized in a series of voltage steps todifferent test potentials and the current in the presence and absence of1 μM Bay K 8644 was recorded. The inward barium current wassignificantly enhanced in magnitude by the addition of Bay K 8644. Thelargest inward barium current (˜160 pA) was recorded when the membranewas depolarized to 0 mV in the presence of 1 μM Bay K 8644. A comparisonof the I-V curves, generated by plotting the largest current recordedafter each depolarization versus the depolarization voltage,corresponding to recordings conducted in the absence and presence of BayK 8644 illustrated the enhancement of the voltage-activated current inthe presence of Bay K 8644.

Pronounced tail currents were detected in the tracings of currentsgenerated in the presence of Bay K 8644 in HEK 293 cells transfectedwith α₁, α₂ and β₁ subunit-encoding cDNA and the lacZ gene, indicatingthat the recombinant calcium channels responsible for thevoltage-activated barium currents recorded in this transfected appear tobe DHP-sensitive.

The second of the two transfected cells that displayed inward bariumcurrents expressed a ˜50 pA current when the membrane was depolarizedfrom −90 mV. This current was nearly completely blocked by 200 μMcadmium, an established calcium channel blocker.

Ten cells that were transiently transfected with the DNA encoding the α₁subunit and the lacZ gene were analyzed by whole-cell patch clampmethods two days after transfection. One of these cells displayed a 30pA inward barium current. This current amplified 2-fold in the presenceof 1 μM Bay K 8644. Furthermore, small tail currents were detected inthe presence of Bay K 8644. These data indicate that expression of thehuman neuronal calcium channel α_(1D) subunit-encoding cDNA in HEK 293yields a functional DHP-sensitive calcium channel.

3. Analysis of HEK 293 Cells Stably Transfected with DNA Encoding HumanNeuronal Calcium Channel Subunits

Individual stably transfected HEK 293 cells were assayedelectrophysiologically for the presence of voltage-dependent bariumcurrents as described for electrophysiological analysis of transientlytransfected HEK 293 cells (see Example VII.B.2.c). In an effort tomaximize calcium channel activity via cyclic-AMP-dependentkinase-mediated phosphorylation [Pelzer, et al. (1990) Rev. Physiol.Biochem. Pharmacol. 114:107-207], cAMP (Na salt, 250 μM) was added tothe pipet solution and forskolin (10 μM) was added to the bath solutionin some of the recordings. Qualitatively similar results were obtainedwhether these compounds were present or not.

Barium currents were recorded from stably transfected cells in theabsence and presence of Bay K 8644 (1 μM). When the cell was depolarizedto −10 mV from a holding potential of −90 mV in the absence of Bay K8644, a current of approximately 35 pA with a rapidly deactivating tailcurrent was recorded. During application of Bay K 8644, an identicaldepolarizing protocol elicited a current of approximately 75 pA,accompanied by an augmented and prolonged tail current. The peakmagnitude of currents recorded from this same cell as a function of aseries of depolarizing voltages were assessed. The responses in thepresence of Bay K 8644 not only increased, but the entirecurrent-voltage relation shifted about −10 mV. Thus, three typicalhallmarks of Bay K 8644 action, namely increased current magnitude,prolonged tail currents, and negatively shifted activation voltage, wereobserved, clearly indicating the expression of a DHP-sensitive calciumchannel in these stably transfected cells. No such effects of Bay K 8644were observed in untransfected HEK 293 cells, either with or withoutcAMP or forskolin.

C. Use of pCMV-based Vectors and pcDNA1-based Vectors for Expression ofDNA Encoding Human Neuronal Calcium Channel Subunits

1. Preparation of Constructs

To determine if the levels of recombinant expression of human calciumchannel subunit-encoding DNA in host cells could be enhanced by usingpCMV-based instead of pcDNA1-based expression vectors, additionalexpression vectors were constructed. The full-length α_(1D) cDNA frompVDCCIII(A) (see Example II.A.3.d), the full-length α₂ cDNA, containedon a 3600 bp EcoRI fragment from HBCaCHα₂ (see Example IV.B) and afull-length β₁ subunit cDNA from pHBCaCHβ_(1b)RBS(A) (see ExampleIII.B.3) were separately subcloned into plasmid pCMVβgal. PlasmidpCMVβgal was digested with NotI to remove the lacZ gene. The remainingvector portion of the plasmid, referred to as pCMV, was blunt-ended atthe NotI sites. The full-length α₂-encoding DNA and β₁-encoding DNA,contained on separate EcoRI fragments, were isolated, blunt-ended andseparately ligated to the blunt-ended vector fragment of PCMV locatingthe cDNAs between the CMV promoter and SV40 polyadenylation sites inpCMV. To ligate the α_(1D)-encoding cDNA with pCMV, the restrictionsites in the polylinkers immediately 5′ of the CMV promoter andimmediately 3′ of the SV40 polyadenylation site were removed from PCMV.A polylinker was added at the NotI site. The polylinker had thefollowing sequence of restriction enzyme recognition sites:

The α_(1D)-encoding DNA, isolated as a BamHI/XhoI fragment frompVDCCIII(A), was then ligated to XbaII/SalI-digested pCMV to place itbetween the CMV promoter and SV40 polyadenylation site.

Plasmid pCMV contains the CMV promoter as does pcDNA1, but differs frompcDNA1 in the location of splice donor/splice acceptor sites relative tothe inserted subunit-encoding DNA. After inserting the subunit-encodingDNA into pCMV, the splice donor/splice acceptor sites are located 3′ ofthe CMV promoter and 5′ of the subunit-encoding DNA start codon. Afterinserting the subunit-encoding DNA into pcDNA1, the splice donor/spliceacceptor sites are located 3′ of the subunit cDNA stop codon.

2. Transfection of HEK 293 Cells

HEK 293 cells were transiently co-transfected with the α_(1D), α₂ and β₁subunit-encoding DNA in PCMV or with the α_(1D), α₂ and βsubunit-encoding DNA in pcDNA1 (vectors pVDCCIII(A), pHBCaCHα₂A andpHBCaCHβ_(1b)RBS(A), respectively), as described in Example VII.B.1.a.Plasmid pCMVβgal was included in each transfection to as a measure oftransfection efficiency. The results of β-galactosidase assays of thetransfectants (see Example VII.B.2.), indicated that HEK 293 cells weretransfected equally efficiently with pCMV- and pcDNA1-based plasmids.

3. Northern Analysis

Total and polyA⁺ RNA were isolated from the transiently transfectedcells as described in Examples VII.A.2 and VII.B.2.b. Northern blots ofthe RNA were hybridized with the following radiolabeled probes: α_(1D)cDNA, human neuronal calcium channel 2 subunit cDNA and DNA encoding thehuman neuronal calcium channel β₁ subunit. Messenger RNA of sizesexpected for α_(1D), α₂ and β₁ subunit transcripts were detected in alltransfectants. A greater amount of the α_(1D) transcript was present incells that were co-transfected with pCMV-based plasmids then in cellsthat were co-transfected with pcDNA1-based plasmids. Equivalent amountsof α₂ and β₁ subunit transcripts were detected in all transfectants.

D. Expression in Xenopus laevis Oöcytes of RNA Encoding Human NeuronalCalcium Channel Subunits

Various combinations of the transcripts of DNA encoding the humanneuronal α_(1D), α₂ and β₁ subunits prepared in vitro were injected intoXenopus laevis oöcytes. Those injected with combinations that includedα_(1D) exhibited voltage-activated barium currents.

1. Preparation of Transcripts

Transcripts encoding the human neuronal calcium channel α_(1D), α₂ andβ₁ subunits were synthesized according to the instructions of the mCAPmRNA CAPPING KIT (Strategene, La Jolla, Calif. catalog #200350).Plasmids pVDCC III.RBS(A), containing of pcDNA1 and the α_(1D) cDNA thatbegins with a ribosome binding site and the eighth ATG codon of thecoding sequence (see Example III.A.3.d), plasmid pHBCaCHα₁A containingof pcDNA1 and an α₂ subunit cDNA (see Example IV), and plasmidpHBCaCHβ_(1b)RBS(A) containing pcDNA1 and the β₁ DNA lacking intronsequence and containing a ribosome binding site (see Example III), werelinearized by restriction digestion. The α_(1D) cDNA- and α₂subunit-encoding plasmids were digested with XhoI, and the β₁subunit-encoding plasmid was digested with EcoRV. The DNA insert wastranscribed with T7 RNA polymerase.

2. Injection of Oöcytes

Xenopus laevis oocytes were isolated and defolliculated by collagenasetreatment and maintained in 100 mM NaCl, 2 mM KC1, 1.8 mM CaCl₂, 1 mMMgCl₂, 5 mM HEPES, pH 7.6, 20 μg/ml ampicillin and 25 μg/ml streptomycinat 19-25° C. for 2 to 5 days after injection and prior to recording. Foreach transcript that was injected into the oöcyte, 6 ng of the specificmRNA was injected per cell in a total volume of 50 nl.

3. Intracellular Voltage Recordings

Injected oöcytes were examined for voltage-dependent barium currentsusing two-electrode voltage clamp methods [Dascal, N. (1987) CRC Crit.Rev. Biochem. 22:317]. The pClamp (Axon Instruments) software packagewas used in conjunction with a Labmaster 125 kHz data acquisitioninterface to generate voltage commands and to acquire and analyze data.Quattro Professional was also used in this analysis. Current signalswere digitized at 1-5 kHz, and filtered appropriately. The bath solutioncontained of the following: 40 mM BaCl₂, 36 mM tetraethylammoniumchloride (TEA-Cl), 2 mM KCl, 5 mM 4-aminopyridine, 0.15 mM niflumicacid, 5 mM HEPES, pH 7.6.

a. Electrophysiological Analysis of Oöcytes Injected with TranscriptsEncoding the Human Neuronal Calcium Channel α₁, α₂ and β₁-subunits

Uninjected oöcytes were examined by two-electrode voltage clamp methodsand a very small (25 nA) endogenous inward Ba²⁺ current was detected inonly one of seven analyzed cells.

Oöcytes coinjected with α_(1D), α₂ and β₁ subunit transcripts expressedsustained inward barium currents upon depolarization of the membranefrom a holding potential of −90 mV or −50 mV (154±129 nA, n=21). Thesecurrents typically showed little inactivation when test pulses rangingfrom 140 to 700 msec. were administered. Depolarization to a series ofvoltages revealed currents that first appeared at approximately −30 mVand peaked at approximately 0 mV.

Application of the DHP Bay K 8644 increased the magnitude of thecurrents, prolonged the tail currents present upon repolarization of thecell and induced a hyperpolarizing shift in current activation. Bay K8644 was prepared fresh from a stock solution in DMSO and introduced asa 10× concentrate directly into the 60 μl bath while the perfusion pumpwas turned off. The DMSO concentration of the final diluted drugsolutions in contact with the cell never exceeded 0.1%. Controlexperiments showed that 0.1% DMSO had no effect on membrane currents.

Application of the DHP antagonist nifedipine (stock solution prepared inDMSO and applied to the cell as described for application of Bay K 8644)blocked a substantial fraction (91±6%, n=7) of the inward barium currentin oöcytes coinjected with transcripts of the α_(1D), α₂ and β₁subunits. A residual inactivating component of the inward barium currenttypically remained after nifedipine application. The inward bariumcurrent was blocked completely by 50 μM Cd²⁺, but only approximately 15%by 100 μM Ni²⁺.

The effect of ωCgTX on the inward barium currents in oöcytes co-injectedwith transcripts of the α_(1D), α₂, and β₁ subunits was investigated.ωcgTX (Bachem, Inc., Torrance Calif.) was prepared in the 15 mM BaCl₂bath solution plus 0.1% cytochrome C (Sigma) to serve as a carrierprotein. Control experiments showed that cytochrome C had no effect oncurrents. A series of voltage pulses from a −90 mV holding potential to0 mV were recorded at 20 msec. intervals. To reduce the inhibition ofωCgTX binding by divalent cations, recordings were made in 15 mM BaCl₂,73.5 mM tetraethylammonium chloride, and the remaining ingredientsidentical to the 40 mM Ba²⁺ recording solution. Bay K 8644 was appliedto the cell prior to addition to ωCgTX in order to determine the effectof ωCgTX on the DHP-sensitive current component that was distinguishedby the prolonged tail currents. The inward barium current was blockedweakly (54±29%, n=7) and reversibly by relatively high concentrations(10-15 μM) of ωCgTX. The test currents and the accompanying tailcurrents were blocked progressively within two to three minutes afterapplication of ωCgTX, but both recovered partially as the ωCgTX wasflushed from the bath.

b. Analysis of Oöcytes Injected with Only a Transcripts Encoding theHuman Neuronal Calcium Channel α_(1D) or Transcripts Encoding an α_(1D)and Other Subunits

The contribution of the α₂ and β₁ subunits to the inward barium currentin oöcytes injected with transcripts encoding the α_(1D), α₂ and β₁subunits was assessed by expression of the α_(1D) subunit alone or incombination with either the β₁ subunit or the α₂ subunit. In oöcytesinjected with only the transcript of a α_(1D) cDNA, no Ba²⁺ currentswere detected (n=3). In oöcytes injected with transcripts of α_(1D) andβ₁ cDNAs, small (108±39 nA) Ba²+ currents were detected upondepolarization of the membrane from a holding potential of −90 mV thatresembled the currents observed in cells injected with transcripts ofα_(1D), α₂ and β₁ cDNAs, although the magnitude of the current was less.In two of the four oöcytes injected with transcripts of theα_(1D)-encoding and β_(1D)-encoding DNA, the Ba²⁺ currents exhibited asensitivity to Bay K 8644 that was similar to the Bay K 8644 sensitivityof Ba²⁺ currents expressed in oöcytes injected with transcripts encodingthe α_(1D) α₁-, α₂₋ and β₁ subunits.

Three of five oöcytes injected with transcripts encoding the α_(1D) andα₂ subunits exhibited very small Ba²⁺ currents (15-30 nA) upondepolarization of the membrane from a holding potential of −90 mV. Thesebarium currents showed little or no response to Bay K 8644.

c. Analysis of Oöcytes Injected with Transcripts Encoding the HumanNeuronal Calcium Channel α₂ and/or β₁ Subunit

To evaluate the contribution of the α_(1D) α₁-subunit to the inwardbarium currents detected in oöcytes co-injected with transcriptsencoding the α_(1D), α₂ and β₁ subunits, oöcytes injected withtranscripts encoding the human neuronal calcium channel α₂ and/or β₁subunits were assayed for barium currents. Oöcytes injected withtranscripts encoding the α₂ subunit displayed no detectable inwardbarium currents (n=5). Oöocytes injected with transcripts encoding a β₁subunit displayed measurable (54±23 nA, n=5) inward barium currents upondepolarization and oöcytes injected with transcripts encoding the α₂ andβ₁ subunits displayed inward barium currents that were approximately 50%larger (80±61 nA, n=18) than those detected in oöcytes injected withtranscripts of the β₁-encoding DNA only.

The inward barium currents in oöcytes injected with transcripts encodingthe β₁ subunit or α₂ and β₁ subunits typically were first observed whenthe membrane was depolarized to −30 mV from a holding potential of −90mV and peaked when the membrane was depolarized to 10 to 20 mV.Macroscopically, the currents in oöcytes injected with transcriptsencoding the α₂ and β₁ subunits or with transcripts encoding the β₁subunit were indistinguishable. In contrast to the currents in oöcytesco-injected with transcripts of α_(1D), α₂ and β₁ subunit cDNAs, thesecurrents showed a significant inactivation during the test pulse and astrong sensitivity to the holding potential. The inward barium currentsin oöcytes co-injected with transcripts encoding the α₂ and β₁ subunitsusually inactivated to 10-60% of the peak magnitude during a 140-msecpulse and were significantly more sensitive to holding potential thanthose in oöcytes co-injected with transcripts encoding the α_(1D), α₂and β₁ subunits. Changing the holding potential of the membranes ofoöcytes co-injected with transcripts encoding the α₂ and β₁ subunitsfrom −90 to −50 mV resulted in an approximately 81% (n=11) reduction inthe magnitude of the inward barium current of these cells. In contrast,the inward barium current measured in oöcytes co-injected withtranscripts encoding the α_(1D), α₂ and β₁ subunits were reducedapproximately 24% (n=11) when the holding potential was changed from −90to −50 mV.

The inward barium currents detected in oöcytes injected with transcriptsencoding the α₂ and β₁ subunits were pharmacologically distinct fromthose observed in oöcytes co-injected with transcripts encoding theα_(1D), α₂ and β₁ subunits. Oöcytes injected with transcripts encodingthe α₂ and β₁ subunits displayed inward barium currents that wereinsensitive to Bay K 8644 (n=11). Nifedipine sensitivity was difficultto measure because of the holding potential sensitivity of nifedipineand the current observed in oöcytes injected with transcripts encodingthe α₂ and β₁ subunits. Nevertheless, two oöcytes that were co-injectedwith transcripts encoding the α₂ and β₁ subunits displayed measurable(25 to 45 nA) inward barium currents when depolarized from a holdingpotential of −50 mV. These currents were insensitive to nifedipine (5 to10 μM). The inward barium currents in oöcytes injected with transcriptsencoding the α₂ and β₁ subunits showed the same sensitivity to heavymetals as the currents detected in oöcytes injected with transcriptsencoding the α_(1D), α₂ and β₁ subunits.

The inward barium current detected in oöcytes injected with transcriptsencoding the human neuronal α₂ and β₁ subunits has pharmacological andbiophysical properties that resemble calcium currents in uninjectedXenopus oöcytes. Because the amino acids of this human neuronal calciumchannel β₁ subunit lack hydrophobic segments capable of formingtransmembrane domains, it is unlikely that recombinant β₁ subunits alonecan form an ion channel. It is more probable that a homologousendogenous α₁ subunit exists in oöcytes and that the activity mediatedby such an α₁ subunit is enhanced by expression of a human neuronal β₁subunit.

E. Expression of DNA Encoding Human Neuronal Calcium Channel α_(1B),α_(2B) and β₁₋₂ Subunits in HEK Cells

1. Transfection of HEK Cells

The transient expression of the human neuronal α_(1B-1), α_(2b) and β₁₋₂subunits was studied in HEK293 cells. The HEK293 cells were grown as amonolayer culture in Dulbecco's modified Eagle's medium (Gibco)containing 5% defined-supplemented bovine calf serum (Hyclone) pluspenicillin G (100 U/ml) and steptomycin sulfate (100 μg/ml). HEK293 celltransfections were mediated by calcium phosphate as described above.Transfected cells were examined for inward Ba²⁺ currents (I_(Ba))mediated by voltage-dependent Ca²⁺ channels.

Cells were transfected (2×10⁶ per polylysine-coated plate. Standardtransfections (10-cm dish) contained 8 μg of pcDNAα_(1B-1), 5 μg ofpHBCaCHα₂A, 2 μg pHBCaCHβ_(1b)RBS(A) (see, Examples II.A.3, IV.B. andIII) and 2 μg of CMVβ (Clontech) β-glactosidase expression plasmid, andpUC18 to maintain a constant mass of 20 μg/ml. Cells were analyzed 48 to72 hours after transfection. Transfection efficiencies (±10%), whichwere determined by in situ histochemical staining for β-galactosidaseactivity (Sanes et al. (1986) EMBO J., 5:3133), generally were greaterthan 50%.

2. Electrophysiological Analysis of Transfectant Currents

1. Materials and Methods

Properties of recombinantly expressed Ca²⁺ channels were studied bywhole cell patch-clamp techniques. Recordings were performed ontransfected HEK293 cells 2 to 3 days after transfection. Cells wereplated at 100,000 to 300,000 cells per polylysine-coated, 35-mm tissueculture dishes (Falcon, Oxnard, Calif.) 24 hours before recordings.Cells were perfused with 15 mM BaCl₂, 125 mM choline chloride, 1 mMMgCl₂, and 10 mM Hepes (pH=7.3) adjusted with tetraethylammoniumhydroxide (bath solution). Pipettes were filled with 135 mM CsCl, 10 mMEGTA, 10 mM Hepes, 4 mM Mg-adenosine triphosphate (pH=7.5) adjusted withtetraethylammonium hydroxide. Sylgard (Dow-Corning, Midland,Mich,)-coated, fire-polished, and filled pipettes had resistances of 1to 2 megohm before gigohm seals were established to cells.

Bay K 8644 and nifedipine (Research Biochemicals, Natick, Mass.) wereprepared from stock solutions (in dimethyl sulfoxide) and diluted intothe bath solution. The dimethyl sulfoxide concentration in the finaldrug solutions in contact with the cells never exceeded 0.1%. Controlexperiments showed that 0.1% dimethyl sulfoxide had no efect on membranecurrents. ωCgTX (Bachem, Inc., Torrance Calif.) was prepared in the 15mM BaCl₂ bath solution plus 0.1% cytochrome C (Sigma, St. Louis Mo.) toserve as a carrier protein. Control experiments showed that cytochrome Chad no effect on currents. These drugs were dissolved in bath solution,and continuously applied by means of puffer pipettes as required for agiven experiment. Recordings were performed at room temperature (22° to25° C.). Series resistance compensation (70 to 85%) was employed tominimize voltage error that resulted from pipette access resistance,typically 2 to 3.5 megohm. Current signals were filtered (−3 dB, 4-poleBessel) at a frequency of 1/4 to 1/5 the sampling rate, which rangedfrom 0.5 to 3 kHz. Voltage commands were generated and data wereacquired with CLAMPEX (pClamp, Axon Instruments, Foster City, Calif.).All reported data are corrected for linear leak and capacitivecomponents. Exponential fitting of currents was performed with CLAMPFIT(Axon Instruments, Foster City, Calif.).

2. Results

Transfectants were examined for inward Ba²⁺ currents (I_(Ba)). Cellscotransfected with DNA encoding α_(1B-1), α_(2B), and β₁₋₂ subunitsexpressed high-voltage-activated Ca²⁺ channels. I_(Ba) first appearedwhen the membrane was depolarized from a holding potential of −90 mV to−20 mV and peaked in magnitude at 10 mV. Thirty-nine of 95 cells (12independent transfections) had I_(Ba) that ranged from 30 to 2700 pA,with a mean of 433 pA. The mean current density was 26 pA/pF, and thehighest density was 150 pA/pF. The I_(Ba) typically increased by 2- to20-fold during the first 5 minutes of recording. Repeateddepolarizations during long records often revealed rundown of I_(Ba)usually not exceeding 20% within 10 min. I_(Ba) typically activatedwithin 10 ms and inactivated with both a fast time constant ranging from46 to 105 ms and a slow time constant ranging from 291 to 453 ms (n=3).Inactivation showed a complex voltage dependence, such that I_(Ba)elicited at ≧20 mV inactivated more slowly than I_(Ba) elicited at lowertest voltages, possibly a result of an increase in the magnitude of slowcompared to fast inactivation components at higher test voltages.

Recombinant α_(1B-1)α_(2b)β₁₋₂ channels were sensitive to holdingpotential. Steady-state inactivation of I_(Ba), measured after a 30- to60-s conditioning at various holding potentials, was approximately 50%at holding potential between −60 and −70 mV and approximately 90% at −40mV. Recovery of I_(Ba) from inactivation was usually incomplete,measuring 55 to 75% of the original magnitude within 1 min. after theholding potential was returned to more negative potentials, possiblyindicating some rundown or a slow recovery rate.

Recombinant α_(1B-1)α_(2b)β₁₋₂ channels were also blocked irreversiblyby ω-CgTx concentrations ranging from 0.5 to 10 μM during the time scaleof the experiments. Application of 5 μM toxin (n=7) blocked the activitycompletely within 2 min., and no recovery of I_(Ba) was observed afterwashing ω-CgTx from the bath for up to 15 min. d²⁺ blockage (50 μM) wasrapid, complete, and reversible; the DHPs Bay K 8644 (1 μM; n=4) ornifedipine (5 μM; n=3) had no discernable effect.

Cells cotransfected with DNA encoding α_(1B-1), α_(2b), β₁₋₂ subunitspredominantly displayed a single class of saturable, high-affinityω-CgTx binding sites. The determined dissociation constant (K_(d)) valuewas 54.6±14.5 pM (n=4). Cells transfected with the vector containingonly β-galactosidase-encoding DNA or α_(2b)β-encoding DNA showed nospecific binding. The binding capacity (B_(max)) of theα_(1B-1)α_(2b)β-transfected cells was 28,710±11,950 sites per cell(n=4).

These results demonstrate that α_(1B-1)α_(2b)β₁₋₂-transfected cellsexpress high-voltage-activated, inactivating Ca²⁺ channel activity thatis irreversibly blocked by ω-CgTx, insensitive to DHPs, and sensitive toholding potential. The activation and inactivation kinetics and voltagesensitivity of the channel formed in these cells are generallyconsistent with previous characterizations of neuronal N-type Ca²⁺channels.

F. Expression of DNA Encoding Human Neuronal Calcium Channel α_(1B-1),α_(1B-2), α_(2B), β₁₋₂ and β₁₋₃ Subunits in HEK Cells

Significant Ba²⁺ currents were not detected in untransfected HEK293cells. Furthermore, untransfected HEK293 cells do not express detectableω-CgTx GVIA binding sites.

In order to approximate the expression of a homogeneous population oftrimeric α_(1B), α_(2b) and β₁ protein complexes in transfected HEK293cells, the α_(1B), α_(2b), β₁ expression levels were altered. Theefficiency of expression and assembly of channel complexes at the cellsurface were optimized by adjusting the molar ratio of α_(1B), α_(2b)and β₁ expression plasmids used in the transfections. The transfectantswere analyzed for mRNA levels, ω-CgTx GVIA binding and Ca²⁺ channelcurrent density in order to determine near optimal channel expression inthe absence of immunological reagents for evaluating protein expression.

1. Transfections

HEK293 cells were maintained in DMEM (Gibco #320-1965AJ), 5.5%Defined/Supplemented bovine calf serum (Hyclone #A-2151-L), 100 U/mlpenicillin G and 100 μg/ml streptomycin. Ca²⁺-phosphate based transienttransfections were performed and analyzed as described above. Cells wereco-transfected with either 8 μg pcDNA1α_(1B-1) (described in ExampleII.C), 5 μg pHBCaCHα₂A (see, Example IV.B.), 2 μg pHBCaCHβ_(1b)RBS(A)(β₁₋₂ expression plasmid; see Examples III.A. and IX.E.), and 2 μgpCMVβ-gal [Clontech, Palo Alto, Calif.] (2:1.8:1 molar ratio of Ca²⁺channel subunit expression plasmids) or with 3 μg pcDNA1α_(1B-1) orpcDNA1α_(1B-2), 11.25 μg pHBCaCHα₂A, 0.75 or 1.0 μg pHBCaCHβ_(1b)RBS(A)or pcDNA1β₁₋₃ and 2 μg pCMVβ-gal (2:10.9:1 molar ratio of Ca²⁺ channelsubunit expression plasmids). Plasmid pCMVβ-gal, a β-galactosidaseexpression plasmid, was included in the transfections as a marker topermit transfection efficiency estimates by histochemical staining. Whenless than three subunits were expressed, pCMVPL2, a pCMVpromoter-containing vector that lacks a cDNA insert, was substituted tomaintain equal moles of pCMV-based DNA in the transfection. pUC18 DNAwas used to maintain the total mass of DNA in the transfection at 20μg/plate.

RNA from the transfected cells was analyzed by Northern blot analysisfor calcium channel subunit mRNA expression using random primed³²P-labeled subunit specific probes. HEK293 cells co-transfected withα_(1B-1), α_(2b) and β₁₋₂ expression plasmids (8, 5 and 2 μg,respectively; molar ratio=2:1.8:1) did not express equivalent levels ofeach Ca²⁺ channel subunit mRNA. Relatively high levels of α_(1B-1) andβ₁₋₂ mRNAs were expressed, but significantly lower levels of α_(2b) mRNAwere expressed. Based on autoradiograph exposures required to produceequivalent signals for all three mRNAs, α_(2b) transcript levels wereestimated to be 5 to 10 times lower than α_(1B-1) and β₁₋₂ transcriptlevels. Untransfected HEK293 cells did not express detectable levels ofα_(1B-1), α_(2b), or β₁₋₂ mRNAs.

To achieve equivalent Ca²⁺ channel subunit mRNA expression levels, aseries of transfections was performed with various amounts of α_(1B-1),α_(2b) and β₁₋₂ expression plasmids. Because the α_(1B-1) and β₁₋₂ mRNAswere expressed at very high levels compared to α_(2b) mRNA, the mass ofα_(1B-1) and β₁₋₂ plasmids was lowered and the mass of α_(2b) plasmidwas increased in the transfection experiments. Co-transfection with 3,11.25 and 0.75 μg of α_(1B-1), α_(2b) and β₁₋₂ expression plasmids,respectively (molar ratio=2:10.9:1), approached equivalent expressionlevels of each Ca²⁺ channel subunit mRNA. The relative molar quantity ofα_(2b) expression plasmid to α_(1B-1) and β₁₋₂ expression plasmids wasincreased 6-fold. The mass of α_(1B-1) and β₁₋₂ plasmids in thetransfection was decreased 2.67-fold and the mass of α_(2b) plasmid wasincreased 2.25-fold. The 6-fold molar increase of α_(2b) relative toα_(1B-1) and β₁₋₂ required to achieve near equal abundance mRNA levelsis consistent with the previous 5- to 10-fold lower estimate of relativeα_(2b) mRNA abundance. ω-CgTx GVIA binding to cells transfected withvarious amounts of expression plasmids indicated that the 3, 11.25 and0.75 μg of α_(1B-1), α_(2b) and β₁₋₂ plasmids, respectively, improvedthe level of cell surface expression of channel complexes. Furtherincreases in the mass of α_(2b) and β₁₋₂ expression plasmids whileα_(1B-1) was held constant, and alterations in the mass of the α_(1B-1)expression plasmid while α_(2b) and β₁₋₂ were held constant, indicatedthat the cell surface expression of ω-CgTx GVIA binding sites per cellwas nearly optimal. All subsequent transfections were performed with 3,11.25 and 0.75 μg or 1.0 μg of α_(1B-1) or α_(1B-2), α_(2b) and β₁₋₂ orβ₁₋₃ expression plasmids, respectively.

2. ¹²⁵I-ω-CgTx GVIA Binding to Transfected Cells

Statistical analysis of the K_(d) and B_(max) values was performed usingone-way analysis of variance (ANOVA) followed by the Tukey-Kramer testfor multiple pairwise comparisons (p≦0.05).

Combinations of human voltage-dependent Ca²⁺ channel subunits, α_(1B-1),α_(1B-2), α_(2b), β₁₋₂ and β₁₋₃, were analyzed for saturation binding of¹²⁵I-ω-CgTx GVIA. About 200,000 cells were used per assay, except forthe α_(1B-1), α_(1B-2), α_(1B-1)α_(2b) and α_(1B-2)α_(2b) combinationswhich were assayed with 1×10⁶ cells per tube. The transfected cellsdisplayed a single-class of saturable, high-affinity binding sites. Thevalues for the dissociation constants (K_(d)) and binding capacities(B_(max)) were determined for the different combinations. The resultsare summarized as follows:

Subunit Combination K_(d) (pM) B_(max) (sites/cell) α_(1B-1)α_(2b)β₁₋₂54.9 ± 11.1 (n = 4) 45,324 ± 15,606 α_(1B-1)α_(2b)β₁₋₃ 53.2 ± 3.6 (n =3) 91,004 ± 37,654 α_(1B-1)β₁₋₂ 17.9 ± 1.9 (n = 3) 5,756 ± 2,163α_(1B-1)β₁₋₃ 17.9 ± 1.6 (n = 3) 8,729 ± 2,980 α_(1B-1)α_(2B) 84.6 ± 15.3(n = 3) 2,256 ± 356   α_(1B-1) 31.7 ± 4.2 (n = 3) 757 ± 128α_(1B-2)α_(2b)β₁₋₂ 53.0 ± 4.8 (n = 3) 19,371 ± 3,798  α_(1B-2)α_(2b)β₁₋₃44.3 ± 8.1 (n = 3) 37,652 ± 8,129  α_(1B-2)β₁₋₂ 16.4 ± 1.2 (n = 3) 2,126± 412   α_(1B-2)β₁₋₃ 22.2 ± 5.8 (n = 3) 2,944 ± 1,168 α_(1B-2)α_(2b)N.D.* (n = 3) N.D. α_(1B-2) N.D. N.D. *N.D. = not detectable

Cells transfected with subunit combinations lacking either the α_(1B-1)or the α_(1B-2) subunit did not exhibit any detectable ¹²⁵I-ωCgTx GVIAbinding (≦600 sites/cell). In addition ¹²⁵I-ω-CgTx GVIA binding toHEK293 cells transfected with α_(1B-2) alone or α_(1B-2)α_(2b) was toolow for reliable Scatchard analysis of the data.

Comparison of the K_(d) and B_(max) values revealed severalrelationships between specific combinations of subunits and the bindingaffinities and capacities of the transfected cells. In cells transfectedwith all three subunits, (α_(1B-1)α_(2b)β₁₋₂-, α_(1B-1)α_(2b)β₁₋₃-,α_(1B-2)α_(2b)β₁₋₂-, or α_(1B-2)α_(2b)β₁₋₃-transfectants) the K_(d)values were indistinguishable (p>0.05), ranging from 44.3±8.1 pM to54.9±11.1 pM. In cells transfected with two-subunit combinations lackingthe α_(2b) subunit (α_(1B-1)β₁₋₂, α_(1B-1)β₁₋₃, α_(1B-2)β₁₋₂ orα_(1B-2)β₁₋₃) the K_(d) values were significantly lower than thethree-subunit combinations (p<0.01), ranging from 16.4±1.2 to 22.2±5.8pM. Cells transfected with only the α_(1B-1) subunit had a K_(d) valueof 31.7±4.2 pM, a value that was not different from the two-subunitcombinations lacking α_(2b) (p<0.05). As with the comparison between thefour α_(1B)α_(2b)β₁ versus α_(1B)β₁ combinations, when the α_(1B-1) wasco-expressed with α_(2b), the K_(d) increased significantly (p<0.05)from 31.7±4.2 to 84.6±5.3 pM. These data demonstrate that co-expressionof the α_(2b) subunit with α_(1B-1), α_(1B-1)β₁₋₂, α_(1B-1)β₁₋₃,α_(1B-2)β₁₋₂ or α_(1B-2)β₁₋₃ subunit combinations results in lowerbinding affinity of the cell surface receptors for ¹²⁵I-ω-CgTx GVIA. TheB_(max) values of cells transfected with various subunit combinationsalso differed considerably. Cells transfected with the α_(1B-1) subunitalone expressed a low but detectable number of binding sites(approximately 750 binding sites/cell). When the α_(1B-1) subunit wasco-expressed with the α_(2b) subunit, the binding capacity increasedapproximately three-fold while co-expression of a β₁₋₂ or β₁₋₃ subunitwith α_(1B-1) resulted in 8- to 10-fold higher expression of surfacebinding. However, cells transfected with all three subunits expressedthe highest number of cell surface receptors. The binding capacities ofcells transfected with α_(1B-1)α_(2b)β₁₋₃ or α_(1B-2)α_(2b)β₁₋₃combinations were approximately two-fold higher than the correspondingcombinations containing the β₁₋₂ subunit. Likewise, cells transfectedwith α_(1B-1)α_(2b)β₁₋₂ or α_(1B-1)α_(2b)β₁₋₃ combinations expressedapproximately 2.5-fold more binding sites per cell than thecorresponding combinations containing α_(1B-2). In all cases,co-expression of the α_(2b) subunit with α_(1B) and β₁ increased thesurface receptor density compared to cells transfected with only thecorresponding α_(1B) and β₁ combinations; approximately 8-fold forα_(1B-1)α_(2b)β₁₋₂, 10-fold for α_(1B-1)α_(2b)β₁₋₃, 9-fold forα_(1B-2)α_(2b)β₁₋₂, and 13-fold for α_(1B-2)α_(2b)β₁₋₃. Thus, insummary, the comparison of the B_(max) values suggests that thetoxin-binding subunit, α_(1B-1) or α_(1B-2), is more efficientlyexpressed and assembled on the cell surface when co-expressed witheither the α_(2b) or the β₁₋₂ or β₁₋₃ subunit, and most efficientlyexpressed when both α_(2b) and β₁ subunits are present.

3. Electrophysiology

Functional expression of α_(1B-1)α_(2b)β₁₋₂ and α_(1B-1)β₁₋₂ subunitcombinations was evaluated using the whole-cell recording technique.Transfected cells that had no contacts with surrounding cells and simplemorphology were used approximately 48 hours after transfection forrecording. The pipette solution was (in mM) 135 CsCl, 10 EGTA, 1 MgCl₂,10 HEPES, and 4 mM Mg-ATP (pH 7.3, adjusted with TEA-OH). The externalsolution was (in MM) 15 BaCl₂, 125 Choline Cl, 1 MgCl₂, and 10 HEPES (pH7.3, adjusted with TEA-OH). ω-CgTx GVIA (Bachem) was prepared in theexternal solution with 0.1% cytochrome C (Sigma) to serve as a carrier.Control experiments showed that cytochrome C had no effect on the Ba²⁺current.

The macroscopic electrophysiological properties of Ba²⁺ currents incells transfected with various amounts of the α_(2b) expression plasmidwith the relative amounts of α_(1B-1) and β₁₋₂ plasmids held constantwere examined. The amplitudes and densities of the Ba²⁺ currents (15 mMBaCl₂) recorded from whole cells of these transfectants differeddramatically. The average currents from 7 to 11 cells of three types oftransfections (no α_(2b) ; 2:1.8:1 [α_(1B-1):α_(2b):β₁₋₂] molar ratio;and 2:10.9:1 [α_(1B-1):α_(2b):β₁₋₂] molar ratio) were determined. Thesmallest currents (range: 10 to 205 pA) were recorded when α_(2b) wasnot included in the transfection, and the largest currents (range: 50 to8300 pA) were recorded with the 2:10.9:1 ratio of α_(1B-1)α_(2b)β₁₋₂plasmids, the ratio that resulted in near equivalent mRNA levels foreach subunit transcript. When the amount of α_(2b) plasmid was adjustedto yield approximately an equal abundance of subunit mRNAs, the averagepeak Ba²⁺ current increased from 433 pA to 1,824 pA (4.2-fold) with acorresponding increase in average current density from 26 pA/pF to 127pA/pF (4.9-fold). This increase is in the presence of a 2.7-folddecrease in the mass of α_(1B-1) and β₁₋₂ expression plasmids in thetransfections. In all transfections, the magnitudes of the Ba²⁺ currentsdid not follow a normal distribution.

To compare the subunit combinations and determine the effects of α_(2b),the current-voltage properties of cells transfected with α_(1B-1)β₁₋₂ orwith α_(1B-1)α_(2b)β₁₋₂ in either the 2:1.8:1 (α_(1B-1):α_(2b):β₁₋₂)molar ratio or the 2:10.9:1 (α_(1B-1):α_(2b):β₁₋₂) molar ratiotransfectants were examined. The extreme examples of no α_(2b) and 11.25μg α_(2b) (2:10.9:1 molar ratio) showed no significant differences inthe current voltage plot at test potentials between 0 mV and +40 mV(p<0.05). The slight differences observed at either side of the peakregion of the current voltage plot were likely due to normalization. Thevery small currents observed in the α_(1B-1)β₁₋₂ transfected cells havea substantially higher component of residual leak relative to the bariumcurrent that is activated by the test pulse. When the current voltageplots are normalized, this leak is a much greater component than in theα_(1B-1)α_(2b)β₁₋₂ transfected cells and as a result, thecurrent-voltage plot appears broader. This is the most likelyexplanation of the apparent differences in the current voltage plots,especially given the fact that the current-voltage plot for theα_(1B-1)β₁₋₂ transfected cells diverge on both sides of the peak.Typically, when the voltage-dependence activation is shifted, the entirecurrent-voltage plot is shifted, which was not observed. Toqualitatively compare the kinetics of each, the average responses oftest pulses from −90 mV to 10 mV were normalized and plotted. Nosignificant differences in activation or inactivation kinetics ofwhole-cell Ba²⁺ currents were observed with any combination.

G. Expression of DNA Encoding Human Neuronal Calcium Channelα_(1E-3)α_(2B)β₁₋₃ and α_(1E-1)α_(2B)β₁₋₃ Subunits in HEK Cells

Functional expression of the α_(1E-1)α_(2B)β₁₋₃ and α_(1E-3)α_(2B)β₁₋₃,as well as α_(1E-1) was evaluated using the whole cell recordingtechnique.

1. Methods

Recordings were performed on transiently transfected HEK 293 cells twodays following the transfection, from cells that had no contacts withsurrounding cells and which had simple morphology.

The internal solution used to fill pipettes for recording the bariumcurrent from the transfected recombinant calcium channels was (in mM)135 CsCl, 10 EGTA, 1 MgCl₂, 10 HEPES, and 4 mM Mg-ATP (pH 7.3, adjustedwith TEA-OH). The external solution for recording the barium current was(in mM) 15 BaCl₂, 125 Choline Cl, 1 MgCl₂, and 10 HEPES (pH 7.3,adjusted with TEA-OH). In experiments in which Ca²⁺ was replaced forBa²⁺, a Laminar flow chamber was used in order to completely exchangethe extracellular solution and prevent any mixing of Ba²⁺ and Ca²⁺.ω-CgTx GVIA was prepared in the external solution with 0.1% cytochrome Cto serve as a carrier, the toxin was applied by pressurized pufferpipette. Series resistance was compensated 70-85% and currents wereanalyzed only if the voltage error from series resistance was less than5 mV. Leak resistance and capacitance was corrected by subtracting thescaled current observed with the P/-4 or -6 protocol as implemented bypClamp (Axon Instruments).

2. Results

a. Electrophysiology

Cells transfected with α_(1E-1)α_(2b)β₁₋₃ or α_(1E-3)α_(2b)β₁₋₃ showedstrong barium currents with whole cell patch clamp recordings. Cellsexpressing α_(1E-3)α_(2B)β₁₋₃ had larger peak currents than thoseexpressing α_(1E-1)α_(2b)β₁₋₃. In addition, the kinetics of activationand inactivation are clearly substantially faster in the cellsexpressing α_(1E) calcium channels. HEK 293 cells expressing α_(1E-1) orα_(1E-3) alone have a significant degree of functional calcium channels,with properties similar to those expressing α_(1E)α_(2b)β₁₋₃ but withsubstantially smaller peak barium currents. Thus, with α_(1E), the α₂and β₁ subunits are not required for functional expression of α_(1E)mediated calcium channels, but do substantially increase the number offunctional calcium channels.

Examination of the current voltage properties of α_(1E)α_(2b)β₁₋₃expressing cells indicates that α_(1E-3)α_(2b)β₁₋₃ is a high-voltageactivated calcium channel and the peak current is reached at a potentialonly slightly less positive than other neuronal calcium channels alsoexpressing α_(2b) and β₁, and α_(1B) and α_(1D). Current voltageproperties of α_(1E-1)α_(2b)β₁₋₃ are statistically different from thoseof α_(1B-1)α_(2b)β₁₋₃. Current voltage curves for α_(1E-1)α_(2b)β₁₋₃ andα_(1E-3)α_(2b)β₁₋₃ both peak at approximately +5 mV, as does the currentvoltage curve for α_(1E-3) alone.

The kinetics and voltage dependence of inactivation using both prepulse(200 ms) and steady-state inactivation was examined. α_(1E) mediatedcalcium channels are rapidly inactivated relative to previously clonedcalcium channels and other high voltage-activated calcium channels.α_(1E-3)α_(2b)β₁₋₃ mediated calcium channels are inactivated rapidly andare thus sensitive to relatively brief (200 ms) prepulses as well aslong prepulses (>20 s steady state inactivation), but recover rapidlyfrom steady state inactivation. The kinetics of the rapid inactivationhas two components, one with a time constant of approximately 25 ms andthe other approximately 400 ms.

To determine whether α_(1E) mediated calcium channels have properties oflow voltage activated calcium channels, the details of tail currentsactivated by a test pulse ranging −60 to +80 mV were measured at −60 mV.Tail currents recorded at −60 mV could be well fit by a singleexponential of 150 to 300 μs; at least an order of magnitude faster thanthose typically observed with low voltage-activated calcium channels.

HEK 293 cells expressing α_(1E-3)α_(2b)β₁₋₃ flux more current with Ba²⁺as the charge carrier and currents carried by Ba²⁺ and Ca²⁺ havedifferent current-voltage properties. Furthermore, the time course ofinactivation is slower and the amount of prepulse inactivation less withCa²⁺ as the charge carrier.

b. Pharmacology

The current of cells transfected with α_(1E-1)α_(2b)β₁₋₃ andα_(1E-3)α_(2b)β₁₋₃ showed sensitivity to both organic and inorganiccalcium channel blockers. Maximal blocking was observed with thenon-specific calcium channel blocker, inorganic Cd²⁺, which reversiblyblocked 95±2% (n=30) of the barium current. A reversible block wasobserved with 50 μM Ni²⁺ of 65±10% (α_(1E-1)α_(2b)β₁₋₃, n=3) to 74±7%(α_(1E-3)α_(2b)β₁₋₃, n=3). In addition, blocking was observed with 300μM amiloride (66±18%, n=4) and ethosuximide (67±10%, n=3). A highsensitivity was observed with ω-Aga-IVa (73±2% block at 10 nM inα_(1E-3)α_(2b)β₁₋₃, n=3) and block by Bay K 8644 (69±3% inα_(1E-3)α_(2b)β₁₋₃ at 5 μM, n=3). Bay K 8644 had no effect on the timecourse of the tail current and a slowing of the inactivation during thetest pulse. Little reversal of the block by either Bay K 8644 orω-Aga-IVa was observed even following the application of brief stronglydepolarizing pulses. Further, Bay K 8644 applied to HEK 293 cellstransfected with α_(1E) alone resulted in 56±6% (n=3) block. Funnel webspider toxin (nFTX, 1:500) resulted in 78±0% in α_(1E-3)α_(2b)β₁₋₃.Little sensitivity was observed to synthetic FTX or the conus snailtoxin ω-CgTx GVIA.

While the invention has been described with some specificity,modifications apparent to those with ordinary skill in the art may bemade without departing from the scope of the invention. Since suchmodifications will be apparent to those of skill in the art, it isintended that this invention be limited only by the scope of theappended claims.

1. An isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule that encodes an α1_(E)-subunit of a human calcium channel and comprises the coding portion of the sequence of nucleotides set forth in SEQ ID NO: 24; (b) a nucleic acid molecule that encodes an α1_(E) subunit of a human calcium channel and comprises the coding portion of the sequence of nucleotides set forth in SEQ ID NO: 27; (c) a nucleic acid molecule that encodes an α_(1E) subunit of a human calcium channel and comprises the sequence of nucleotides set forth in SEQ ID NO: 25, wherein the encoded α1_(E) subunit has a molecular weight greater than about 120 kilodaltons (kD) and is a full-length α1_(E) subunit of a human calcium channel that can form an ion channel; (d) a nucleic acid molecule comprising a sequence of nucleotides with codons that are degenerate to the codons in the coding portion of the sequence of nucleotides set forth in (a) or (b) above; (e) a nucleic acid molecule comprising a sequence of nucleotides that encodes an α1_(E) subunit of a human calcium channel that comprises a sequence of amino acid sequence encoded by the nucleic acid molecule of any one of (a), (b), (c) or (d) above; and (f) a nucleic acid molecule that encodes an α1_(E) subunit of a human calcium channel polypeptide, wherein the nucleic acid molecule hybridizes under stringent wash condition, 0.1.times.SSC, 0.1% SDS at 65° C., to the complement of the nucleotide sequence of (a), (b), (c), (d) or (e) above.
 2. The isolated nucleic acid molecule of claim 1, wherein the subunit is an α_(1E) subunit or α_(1E-3)-subunit.
 3. The isolated nucleic acid molecule of claim 2, wherein the subunit is an α_(1E-1)-subunit.
 4. The isolated nucleic acid molecule of claim 1, wherein the α₁ subunit is an α_(1E-3) subunit.
 5. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit of a human calcium channel, wherein the α_(1E)-subunit has an amino sequence of a subunit encoded by the isolated nucleic acid molecule of claim
 1. 6. The eukaryotic cell of claim 5, wherein the heterologous nucleic acid molecule is a DNA molecule, wherein the DNA molecule encodes an α_(1E-1)-subunit of a human calcium channel.
 7. The eukaryotic cell of claim 5, wherein the heterologous nucleic acid molecule is a DNA molecule, wherein the DNA molecule encodes an α_(1E-3)-subunit of a human calcium channel.
 8. The eukaryotic cell of claim 5, wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 9. The eukaryotic cell of claim 6 wherein said cell that expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 10. The eukaryotic cell of claim 7 wherein said cell that expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 11. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the eukaryotic cell of claim 8 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membranes of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is (I) not exposed to the test compound or the control cell is identical to the cell of claim 8 except that the control cell does not express functional calcium channels.
 12. The cell of claim 11 selected from the group consisting of an HEK 293 cell, a Chinese hamster ovary cell, an African green monkey cell, and a mouse L cell.
 13. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 9 a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 9 except that the control cell does not express functional calcium channels.
 14. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 10 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 10 except that the control cell does not express functional calcium channels.
 15. A recombinant eukaryotic cell that expresses a functional, heterologous calcium channel, which is produced by a process comprising (a) introducing into suitable host cells an RNA transcript encoding an α_(1E)-subunit of a human calcium channel; (b) culturing and harvesting the host cells of step (a) under conditions favoring expression of the α_(1E)-subunit of a human calcium channels in said cell; and (c) isolating said cell, wherein: the α₁-subunit has an amino sequence of a subunit encoded by the nucleic acid molecule of claim 1; the heterologous calcium channels are the only heterologous ion channels expressed by the cell; and the cell is an amphibian oöcyte.
 16. The eukaryotic cell of claim 15, wherein: the process further comprises introducing a second RNA that is translatable in the cell into an α₂-subunit of a calcium channel.
 17. The eukaryotic cell of claim 15, wherein: the calcium channels also comprise a β₁-subunit of a calcium channel.
 18. The eukaryotic cell of claim 17, wherein: the calcium channels also comprise a α₂-subunit of a calcium channel.
 19. The eukaryotic cell of claim 5, further comprising a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α₂-subunit of a calcium channel.
 20. The eukaryotic cell of claim 5, wherein the α1E-subunit is an α_(1E-1)-subunit of a human calcium channel.
 21. The eukaryotic cell of claim 19 which is selected from the group consisting of an HEK 293 cell, a Chinese hamster ovary cell, an African green monkey cell and a mouse L cell.
 22. The eukaryotic cell of claim 15, wherein: the process further comprises introducing a second RNA that is translatable in the cell into a β₁-subunit of a calcium channel.
 23. The eukaryotic cell of claim 22, wherein: the process further comprises introducing a third mRNA that is translatable in the cell into an α₂-subunit of a human calcium channel.
 24. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 15 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; and detecting the current flowing into the cell.
 25. An expression vector comprising the nucleic acid molecule of claim 1, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 26. The expression vector of claim 25, wherein said vector is a plasmid.
 27. An expression vector comprising the nucleic acid molecule of claim 2, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 28. The expression vector of claim 27, wherein said vector is a plasmid.
 29. An isolated nucleic acid molecule, comprising a sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO.
 24. 30. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E) -subunit of a human calcium channel, wherein the α_(1E)-subunit is encoded by the isolated nucleic acid molecule of claim
 29. 31. The eukaryotic cell of claim 30 wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 32. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 31 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 31 except that the control cell does not express functional calcium channels.
 33. A recombinant eukaryotic cell that expresses a functional, heterologous calcium channel, which is produced by a process comprising (a) introducing into suitable host cells an RNA transcript encoding an α_(1E)-subunit of a human calcium channel; (b) culturing and harvesting the host cells of step (a) under conditions favoring expression of the α_(1E)-subunit of a human calcium channels in said cell; and (c) isolating said cell, wherein: the α₁-subunit has an amino sequence of a subunit encoded by the nucleic acid molecule of claim 29; the heterologous calcium channels are the only heterologous ion channels expressed by the cell; and the cell is an amphibian oöcyte.
 34. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 33 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; and detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 33 except that the control cell does not express functional calcium channels.
 35. An expression vector comprising the nucleic acid molecule of claim 29, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 36. The expression vector of claim 35, wherein said vector is a plasmid.
 37. An isolated nucleic acid molecule, comprising a sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO.
 27. 38. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit of a human calcium channel, wherein the α_(1E)-subunit is encoded by the molecule of claim
 37. 39. The eukaryotic cell of claim 38 wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 40. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 39 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 39 except that the control cell does not express functional calcium channels.
 41. A recombinant eukaryotic cell that expresses a functional, heterologous calcium channel, which is produced by a process comprising (a) introducing into suitable host cells an RNA transcript encoding an α_(1E)-subunit of a human calcium channel; (b) culturing and harvesting the host cells of step (a) under conditions favoring expression of the α_(1E)-subunit of a human calcium channels in said cell; and (c) isolating said cell, wherein: the α₁-subunit has an amino sequence of a subunit encoded by the nucleic acid molecule of claim 37; the heterologous calcium channels are the only heterologous ion channels expressed by the cell; and the cell is an amphibian oöcyte.
 42. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending a cell of claim 41 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein: control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is_identical to the cell of claim 41 except that the control cell does not express functional calcium channels.
 43. An expression vector comprising the nucleic acid molecule of claim 37, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 44. The expression vector of claim 43, wherein said vector is a plasmid.
 45. An isolated nucleic acid molecule, comprising the sequence of nucleotides set forth in nucleotides 169-6921 of SEQ ID No.
 24. 46. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit of a human calcium channel, wherein the α_(1E)-subunit is encoded by the molecule of claim
 45. 47. The eukaryotic cell of claim 46 wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 48. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 47 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current thus detected to a current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 47 except that the control cell does not express functional calcium channels.
 49. An expression vector comprising the nucleic acid molecule of claim 45, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 50. The expression vector of claim 49, wherein said vector is a plasmid.
 51. An isolated nucleic acid molecule that encodes an α_(1E) subunit of a human calcium channel and comprises a sequence of amino acids encoded by the sequence of nucleotides set forth in nucleotides 166-6978 of SEQ ID NO.
 27. 52. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit of a human calcium channel, wherein the α_(1E)-subunit is encoded by the molecule of claim
 51. 53. The eukaryotic cell of claim 52 wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous nucleic acid molecule.
 54. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 53 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell, and wherein: the control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 53 except that the control cell does not express functional calcium channels.
 55. An expression vector comprising the nucleic acid molecule of claim 51, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 56. The expression vector of claim 55, wherein said vector is a plasmid.
 57. An isolated nucleic acid molecule, comprising the sequence of nucleotides set forth in nucleotides 166-6978 of SEQ ID NO.
 27. 58. A eukaryotic cell transfected with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit of a human calcium channel, wherein the α_(1E)-subunit is encoded by the molecule of claim
 57. 59. The eukaryotic cell of claim 58 wherein said cell expresses a functional heterologous calcium channel comprising at least one subunit encoded by the heterologous DNA.
 60. A method for identifying a compound that modulates the activity of a calcium channel, comprising: suspending the cell of claim 59 in a solution containing the compound and a calcium channel selective ion; depolarizing the cell membrane of the cell; and detecting the current flowing into the cell; and comparing the current with the current flowing into a control cell, wherein the current that is detected differs from that detected in the control cell and wherein: control cell is treated substantially the same as the cell exposed to the test compound except that the control culture is not exposed to the test compound or the control cell is identical to the cell of claim 59 except that the control cell does not express functional calcium channels.
 61. An expression vector comprising the nucleic acid molecule of claim 57, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 62. The expression vector of claim 61, wherein said vector is a plasmid.
 63. An isolated nucleic acid molecule that encodes an α_(1E)-subunit of a human calcium channel comprising a sequence of amino acids encoded by the sequence of nucleotides set forth in nucleotides 166-6978 of SEQ ID NO.
 27. 64. A eukaryotic cell transferred with a heterologous nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E)-subunit is encoded by the molecule of claim
 63. 65. An expression vector comprising the nucleic acid molecule of claim 63, operably linked to a regulatory nucleotide sequence that controls expression of the nucleic acid molecule in a host cell.
 66. The expression vector of claim 65, wherein said vector is a plasmid.
 67. An isolated RNA molecule selected from the group consisting of: (a) a nucleic acid molecule that encodes an α1_(E)-subunit of a human calcium channel and comprises the coding portion of the sequence of nucleotides set forth in SEQ ID NO: 24; (b) a nucleic acid molecule that encodes an α1_(E) subunit of a human calcium channel and comprises the coding portion of the sequence of nucleotides set forth in SEQ ID NO: 27; (c) a nucleic acid molecule that encodes an α1_(E) subunit of a human calcium channel and comprises the sequence of nucleotides set forth in SEQ ID NO: 25, wherein the encoded α_(1E) subunit has a molecular weight greater than about 120 kilodaltons (kD) and is a full-length α1_(E) subunit of a human calcium channel that can form an ion channel; (d) a nucleic acid molecule comprising a sequence of nucleotides with codons that are degenerate to the codons in the coding portion of the sequence of nucleotides set forth in (a) or (b) above; (e) a nucleic acid molecule comprising a sequence of nucleotides that encodes an α_(1E) subunit of a human calcium channel that comprises a sequence of amino acid sequence encoded by the nucleic acid molecule of any one of (a), (b), (c) or (d) above; and (f) a nucleic acid molecule that encodes an α1_(E) subunit of a human calcium channel polypeptide, wherein the nucleic acid molecule hybridizes under stringent wash condition, 0.1.times.SSC, 0.1% SDS at 65° C., to the complement of the nucleotide sequence of (a), (b), (c), (d) or (e) above.
 68. An isolated nucleic acid molecule that encodes an _(α1E)-subunit of a human calcium channel, wherein the _(α1E)-subunit can form a functional calcium channel. 